JP7358367B2 - Anti-glycoMUC1 antibody and its use - Google Patents

Description

1. SEQUENCE LISTING This application contains a Sequence Listing submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on October 24, 2017 and is located in GOT-001WO_Sequence_Listing. txt and has a size of 33,265 bytes.

2. Background Human mucin MUC1 is a pleomorphic transmembrane glycoprotein expressed on the apical surface of monolayered and glandular epithelia (Taylor-Papadimitriou et al., 1999). MUC1 is highly overexpressed and aberrantly O-glycosylated in adenocarcinomas. The extracellular domain of mucin contains a variable number of tandem repeats (TR) (25-125) of 20 amino acid residues with five potential sites for O-glycosylation. O-glycans are incompletely processed in cancer cells, resulting in the expression of the pancarcinoma carbohydrate antigen Tn (GalNAcα1-O-Ser/Thr) (Springer, 1984). Tn, a simple mucin-type O-glycan, is widely expressed in adenocarcinomas (including breast and ovarian cancers) and has a restricted distribution in normal adult tissues (Springer, 1984). Expression of these O-glycans in cancer correlates with poor prognosis, and cancer patients have increased natural antibodies against these carbohydrate haptens (Miles et al., 1995; Soares et al., 1996; Werther et al. et al., 1996). There is a need in the art for therapeutic methods that utilize the glycoMUC1 epitope that is overexpressed on cancer cells.

3. Summary The present disclosure captures tumor specificity of glycopeptide variants by providing therapeutic and diagnostic agents based on antibodies and antigen-binding fragments that are selective for cancer-specific epitopes of glycoMUC1.

The present disclosure provides anti-glyco-MUC1 antibodies and antigen-binding fragments thereof that bind to cancer-specific glycosylation variants of MUC1. The present disclosure further provides fusion proteins and antibody-drug conjugates comprising anti-glycoMUC1 antibodies and antigen-binding fragments, and nucleic acids encoding anti-glycoMUC1 antibodies, antigen-binding fragments and fusion proteins.

The present disclosure further provides methods of using anti-glycoMUC1 antibodies, antigen-binding fragments, fusion proteins, antibody-drug conjugates, and nucleic acids for cancer therapy.

In certain aspects, the present disclosure provides bispecific and other multispecific anti-glyco-MUC1 antibodies and antigen-binding fragments that bind cancer-specific glycosylation variants of MUC1 and a second epitope. I will provide a. The second epitope can be on either MUC1 itself, another protein coexpressed with MUC1 on cancer cells, or another protein present on a different cell, such as an activated T cell. . Further disclosed are nucleic acids encoding antibodies, such as nucleic acids that include coding regions that are codon-optimized, and nucleic acids that include coding regions that are not codon-optimized for expression in a particular host cell.

Anti-glycoMUC1 antibodies and binding fragments can be in the form of fusion proteins containing fusion partners. The fusion partner may be useful in providing a second function, such as a signaling function of a signaling domain of a T cell signaling protein, a peptide modulator of T cell activation, or an enzymatic component of a labeling system. . Exemplary T cell signaling proteins include 4-1BB, CO3C, and fusion peptides such as CD28-CD3-zeta and 4-1BB-CD3-zeta. 4-1BB, or CD137, is the costimulatory receptor for T cells, and CD3-zeta is the signaling component of the T cell antigen receptor. The moiety providing the second function may be a modulator of T cell activation, such as IL-15, IL-15Ra, or an IL-15/IL-15Ra fusion, or the extent of binding in vivo or in vitro. and/or may encode a label or enzymatic component of a labeling system useful for location monitoring. Constructs encoding these biomolecules with prophylactic and therapeutic activity when placed in the context of T cells, e.g., autologous T cells, prevent or treat various cancers in some embodiments of the present disclosure. provides a powerful platform for replenishing adoptively transferred T cells.

In certain embodiments, the anti-glycoMUC1 antibodies or antigen-binding fragments of the present disclosure comprise (or are encoded by) the heavy chain and/or light chain variable sequences set forth in Table 1. For clarity, when the term "anti-glycoMUC1 antibody" is used in this document, it refers to monospecific and multispecific (including bispecific) antibodies, unless the context indicates otherwise. It is intended to include anti-glycoMUC1 antibodies, antigen-binding fragments of monospecific and multispecific antibodies, and fusion proteins and conjugates containing antibodies and antigen-binding fragments thereof. Similarly, when the term "anti-glyco-MUC1 antibody or antigen-binding fragment" is used, it also includes monospecific and multispecific (including bispecific) anti-glyco-MUC1 antibodies or antigen-binding fragments, unless the context indicates otherwise. In addition to MUC1 antibodies and antigen-binding fragments thereof, fusion proteins and conjugates containing such antibodies and antigen-binding fragments are also intended to be included.

In other embodiments, the anti-glycoMUC1 antibodies or antigen-binding fragments of the present disclosure comprise (or are encoded by) the heavy chain and/or light chain CDR sequences set forth in Tables 1-3. The CDR sequences listed in Table 1 are used for IMGT (Lefranc et al., 2003, Dev Comparat Immunol 27:55-77, Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological) to define the boundaries of the CDRs. Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.), and CDRs defined according to the scheme of Chothia (Al-Lazikani et al., 1997, J. Mol. Biol 273:927-948). The CDR sequences listed in Table 2 are the combined overlapping regions of the CDR sequences listed in Table 1, with IMGT, Kabat and Chothia sequences shown in underlined bold type. The sequences are the common overlapping regions of the CDR sequences shown in Table 1. The framework sequences of such anti-glycoMUC1 antibodies and antigen binding fragments are the native murine framework sequences listed in Table 1. or non-natural (eg, humanized or human) framework sequences.

In certain aspects, the present disclosure provides that the anti-glycoMUC1 antibodies or antigen-binding fragments of the present disclosure include CDRs that include the amino acid sequence of any of the CDR combinations set forth in numbered embodiments 3 through 17. do. Accordingly, in certain embodiments, the anti-glyco-MUC1 antibodies or antigen-binding fragments of the present disclosure include CDR-H1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, amino acid sequence of SEQ ID NO: 25, CDR-H3 comprising the sequence SEQ ID NO: 8, CDR-L1 comprising the amino acid sequence SEQ ID NO: 8, CDR-L2 comprising the amino acid sequence SEQ ID NO: 9, and CDR-L3 comprising the amino acid sequence SEQ ID NO: 31. In some embodiments, the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 5, 23, 28, or 32. In some embodiments, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6 or 24. In some embodiments, CDR-H3 comprises the amino acid sequence of SEQ ID NO:7. In some embodiments, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 30 or 26. In some embodiments, CDR-L2 comprises the amino acid sequence of SEQ ID NO:27. In some embodiments, CDR-L3 comprises the amino acid sequence of SEQ ID NO: 10. In other embodiments, the anti-glycoMUC1 antibodies or antigen binding fragments of the present disclosure comprise heavy chain CDRs of SEQ ID NOs: 5-7 and light chain CDRs of SEQ ID NOs: 8-10. In other embodiments, the anti-glycoMUC1 antibodies or antigen binding fragments of the present disclosure comprise the heavy chain CDRs of SEQ ID NOs: 23-25 and the light chain CDRs of SEQ ID NOs: 26, 27, and 10. In other embodiments, the anti-glycoMUC1 antibodies or antigen binding fragments of the present disclosure comprise the heavy chain CDRs of SEQ ID NOs: 28, 29, and 25 and the light chain CDRs of SEQ ID NOs: 30, 9, and 31. In other embodiments, the anti-glycoMUC1 antibodies or antigen binding fragments of the present disclosure comprise the heavy chain CDRs of SEQ ID NO: 32, 24, and 7 and the light chain CDRs of SEQ ID NO: 26, 27, and 10. In other embodiments, the anti-glycoMUC1 antibodies or antigen binding fragments of the present disclosure comprise the heavy chain CDRs of SEQ ID NO: 33, 29, and 25 and the light chain CDRs of SEQ ID NO: 8, 9, and 31. The antibody or antigen-binding fragment may be a murine, chimeric, humanized or human antibody or an antigen-binding fragment thereof.

In a further aspect, an anti-glycoMUCl antibody or antigen-binding fragment of the present disclosure competes with an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of SEQ ID NO: 3 and 4, respectively. In yet other embodiments, the present disclosure provides anti-MUC1 antibodies having heavy and light chain variable regions having at least 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NOs: 3 and 4, respectively. or provide an antigen-binding fragment.

In yet other embodiments, the anti-glycoMUC1 antibodies or antigen binding fragments of the present disclosure are single chain variable fragments (scFv). An exemplary scFv includes a heavy chain variable fragment N-terminal to a light chain variable fragment. In some embodiments, the heavy and light chain variable fragments of the scFv are covalently linked to a linker sequence of 4-15 amino acids. The scFv may be in the form of a bispecific T cell derivative or within a chimeric antigen receptor (CAR).

Anti-glycoMUCl antibodies and antigen binding fragments may be in the form of multimers of single chain variable fragments, bispecific single chain variable fragments, and multimers of bispecific single chain variable fragments. In some embodiments, the multimer of single chain variable fragments is selected from divalent single chain variable fragments, tribodies or tetrabodies. In some of these embodiments, the multimer of bispecific single chain variable fragments is a bispecific T cell derivative.

Other aspects of the disclosure are interested in nucleic acids encoding the anti-glycoMUC1 antibodies and antigen-binding fragments of the disclosure. In some embodiments, the portion of the nucleic acid encoding the anti-glycoMUC1 antibody or antigen-binding fragment is codon-optimized for expression in human cells. In certain embodiments, the present disclosure provides heavy chain nucleotide sequences having at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 11 or SEQ ID NO: 13, and SEQ ID NO: 12 or SEQ ID NO: Provided is an anti-glyco-MUC1 antibody or antigen-binding fragment having a heavy chain and light chain variable region encoded by a light chain nucleotide sequence having at least 95%, 98%, 99%, or 99.5% sequence identity with No. 14. do. Vectors containing nucleic acids (eg, viral vectors such as lentiviral vectors) and host cells are also within the scope of this disclosure. The heavy and light chain encoding sequences may be present on a single vector or on separate vectors.

Yet another aspect of the present disclosure provides an anti-glycoMUC1 antibody, an antigen binding fragment, a nucleic acid (or pair of nucleic acids), a vector (or pair of vectors) or a host cell according to the present disclosure, and a physiologically suitable buffer, adjuvant. or a pharmaceutical composition containing a diluent.

Yet another aspect of the present disclosure is a method of making a chimeric antigen receptor comprising: incubating a cell comprising a nucleic acid or vector according to the present disclosure under conditions suitable for expression of a coding region; The method involves collecting the body.

Another aspect of the present disclosure is a method of detecting cancer comprising: contacting a cell or tissue sample with an anti-glycoMUC1 antibody or antigen-binding fragment of the present disclosure, and wherein the antibody binds to the cell or tissue sample. The method includes detecting whether the

Yet another aspect of the present disclosure is a method of treating cancer, comprising administering a prophylactically or therapeutically effective amount of an anti-glycoMUC1 antibody, antigen-binding fragment, nucleic acid, vector, host cell or pharmaceutical composition according to the present disclosure. , to a subject in need thereof.

FIG. 3 shows the results of an ELISA assay showing the binding specificity of GO2 to glycoMUC1 relative to MUC1. FIG. 2 shows the binding of GO2 to colon cancer tissue. Immunohistochemical labeling of invasive colon cancer tissue and adjacent healthy tissue using mAb GO2. The mAb GO2 shows distinct binding to colon cancer tissue and has high reactivity both within the cells and on the surface structures of cancer cells. In contrast, no reactivity to the surface structures of healthy colon cells is seen. FIG. 2 is a diagram showing the binding of GO2 to pancreatic cancer tissue. Immunohistochemical labeling of pancreatic cancer tissue using mAb GO2. mAb GO2 shows distinct binding to pancreatic cancer cells. In contrast, there was no or limited reactivity to surrounding healthy tissue. FIG. 3 shows the binding of GO2 to breast cancer tissue. Immunohistochemical labeling of breast cancer tissue using mAb GO2. mAb GO2 showed distinct binding to invasive breast cancer cells. FIG. 3 shows the results of an antibody-dependent cytotoxicity assay using antibody GO2 and a secondary antibody conjugated to the anti-tubulin agent monomethyl auristatin F (MMAF). FIG. 2 shows the results of an ELISA assay using GO2 to quantify circulating tumor cells. The X-axis shows the number of cells and the Y-axis shows the OD450 value. FIG. 4 shows representative images of MUC1-positive TMA tumor cores. Figure 7A: Breast cancer; Figure 7B: Non-small cell lung cancer; Figure 7C: Ovarian cancer; Figure 7D: Colorectal cancer; Figure 7E: Prostate cancer. 1 is a schematic representation of an exemplary anti-glycoMUC1 and anti-CD3 T cell bispecific antibody (TCB). FIG. 3 shows Jurkat-NFAT activation assay using undigested patient-derived tumor samples (bronchial and pulmonary malignant neoplasms: middle lobe, bronchial or pulmonary, squamous cell carcinoma) and 50 nM of different TCBs. FIG. 3 shows Jurkat-NFAT activation assay using undigested patient-derived tumor samples (bronchial and pulmonary malignant neoplasms: middle lobe, bronchial or pulmonary, squamous cell carcinoma) and 5 nM of different TCBs. In figure showing Jurkat-NFAT activation assay using undigested patient-derived tumor samples (bronchial and pulmonary malignant neoplasms: lower lobe, bronchus or lung, non-keratinizing squamous cell carcinoma) and 50 nM of different TCBs. be. FIG. 3 shows Jurkat-NFAT activation assay using undigested patient-derived tumor samples (bronchial and pulmonary malignant neoplasms: upper lobe, bronchial or pulmonary, adenocarcinoma with acinar type) and 50 nM of different TCBs. . FIG. 3 shows the binding of GO2 TCB to MUC1 expressed on MCF7 cs cells as measured by flow cytometry. FIG. 3 shows the binding of GO2 TCB to MUC1 expressed on T3M4 pzfv cells as measured by flow cytometry. Upregulation of CD25 and CD69 on CD4 and CD8 T cells in the presence of PBMC from two healthy donors, plus IL6, IL8, IL10, IFNγ, TNFα and granzyme by GO2 TCB on T3M4 pzfv FIG. 13A-13L shows tumor cell killing and induction of T cell activation as measured by release of B (Donor 1 FIGS. 13A-13L). The legend is the same for each of Figures 13A-13X. Upregulation of CD25 and CD69 on CD4 and CD8 T cells in the presence of PBMC from two healthy donors, plus IL6, IL8, IL10, IFNγ, TNFα and granzyme by GO2 TCB on T3M4 pzfv FIG. 13A-13L shows tumor cell killing and induction of T cell activation as measured by release of B (Donor 1 FIGS. 13A-13L). The legend is the same for each of Figures 13A-13X. Upregulation of CD25 and CD69 on CD4 and CD8 T cells in the presence of PBMC from two healthy donors, plus IL6, IL8, IL10, IFNγ, TNFα and granzyme by GO2 TCB on T3M4 pzfv FIG. 13 shows the induction of tumor cell killing and T cell activation as measured by the release of B (Donor 2 FIGS. 13M-13X). The legend is the same for each of Figures 13A-13X. Upregulation of CD25 and CD69 on CD4 and CD8 T cells in the presence of PBMC from two healthy donors, plus IL6, IL8, IL10, IFNγ, TNFα and granzyme by GO2 TCB on T3M4 pzfv FIG. 13 shows the induction of tumor cell killing and T cell activation as measured by the release of B (Donor 2 FIGS. 13M-13X). The legend is the same for each of Figures 13A-13X. Tumor cell killing (Figures 14A-14B) and induction of T cell activation as measured by upregulation of CD25 and CD69 on CD8 and CD4 T cells using GO2 TCB on MCF7 cs in the presence of PBMC. (FIGS. 14C to 14F, respectively). The legend is the same for each of Figures 14A-14F. FIG. 15 shows binding of GO2 TCB and HMFG1 TCB to MCF10A (human non-tumorigenic mammary epithelial cell line) (FIG. 15A) and HBEpiC (human bronchial epithelial cell) (FIG. 15B). Tumor cell lethality as measured by upregulation of CD25 on CD4T cells (Figure 16B) and CD8T cells (Figure 16C) by GO2 TCB and HMFG1 TCB on MCF10A cells in the presence of PBMC (Figure 16A) and induction of T cell activation. FIG. 2 is an illustration of GO2 and GO2 TCB flowing through a flow cell with glycopeptides coupled thereto. FIG. 2 is a sensorgram showing the binding of GO2 to human and cynomolgous glycopeptides. FIG. 3 is a sensorgram showing the binding of GO2 TCB to human and cynomolgus monkey glycopeptides. FIGS. 19A-B: GO2 antibody binding (avidity) to human and cynomolgus monkey glycopeptides and estimates of “apparent” KD. Figures 19C-D: Estimated binding of GO2 TCB to human and cynomolgus monkey glycopeptides (avidity) and "apparent" KD.

5. Detailed Description 5.1 Antibodies The inventors have developed novel antibodies directed against the glycoforms of MUC1 presented on tumor cells. These are exemplified by antibody 5F7, referred to herein as "GO2." MUC1 was glycosylated with purified recombinant human glycosyltransferase polypeptides GalNAc-T2, GalNAc-T4, and GalNAc-T1 to mimic the glycosylation pattern of MUC1 presented on tumor cells. GO2 was identified in a screen for antibodies that bind to three representative copies of a glycosylated 60-mer of one of the tandem repeats present in VTSAPDTRPAPGSTAPPAHG (SEQ ID NO: 50).

The anti-glycoMUC1 antibodies of the present disclosure, exemplified by antibody GO2, are useful as tools in cancer diagnosis and therapy.

Accordingly, in certain embodiments, the present disclosure relates to a glycoform of MUC1 (referred to herein as "glycoMUC1") that is presented on tumor cells, preferably as described in U.S. Patent No. 6,465,220. Antibodies and antigen-binding fragments that bind to the 60-mer peptide (VTSAPDTRPAPGSTAPPAHG) ₃ (SEQ ID NO: 47) glycosylated with GalNAc-T2, GalNAc-T4, and GalNAc-T1 are provided herein.

The anti-glycoMUC1 antibodies of the present disclosure may be polyclonal, monoclonal, genetically engineered, and/or otherwise modified, such as, but not limited to, chimeric antibodies, human Examples include primatized antibodies, human antibodies, primatized antibodies, single chain antibodies, bispecific antibodies, dual variable domain antibodies, and the like. In various embodiments, the antibody comprises all or a portion of an antibody constant region. In some embodiments, the constant region is an isotype selected from IgA (e.g., _IgAi or _IgA2 ), IgD, IgE, IgG (e.g., _IgGi , _IgG2 , _IgG3 or _IgG4 ), and IgM. It is. In specific embodiments, the anti-glycoMUC1 antibodies of the present disclosure comprise a constant region isotype of _IgG1 .

The term "monoclonal antibody" as used herein is not limited to antibodies produced via hybridoma technology. Monoclonal antibodies include any eukaryotic, prokaryotic, or phage clone that is derived from a single clone by any means available or known in the art. Monoclonal antibodies useful in disclosing the invention can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or combinations thereof. In many uses of the present disclosure, including in vivo uses of anti-glycoMUC1 antibodies in humans, chimeric, primatized, humanized, or human antibodies can be suitably used.

The term "chimeric" antibody, as used herein, combines variable sequences derived from a non-human immunoglobulin, such as a rat or mouse antibody, and human immunoglobulin constant regions typically selected from a human immunoglobulin template. Refers to antibodies that have Methods for producing chimeric antibodies are known in the art. For example, Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies et al., 1985, incorporated herein by this reference in their entirety. , J. Immunol. Methods 125:191-202; US Pat. No. 5,807,715; US Pat. No. 4,816,567; and US Pat. No. 4,816,397.

A "humanized" form of a non-human (eg, murine) antibody is a chimeric immunoglobulin that contains minimal sequence derived from a non-human immunoglobulin. Generally, a humanized antibody has all or substantially all of its CDR regions corresponding to CDR regions of a non-human immunoglobulin and all or substantially all of its FR regions being FR regions of human immunoglobulin sequences. It is expected to include substantially all of at least one, typically two, variable domains. A humanized antibody may include at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin consensus sequence. Methods of antibody humanization are known in the art. See, for example, Riechmann et al., 1988, Nature 332:323-7; United States Patent No. 5,530,101 to Queen et al.; No. 5,585, all of which are incorporated herein by reference in their entirety. ,089; 5,693,761; 5,693,762; and 6,180,370; European Patent No. 239400; PCT International Publication No. 91 /09967 pamphlet; US Patent No. 5,225,539; European Patent No. 592106; European Patent No. 519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci. 91:969-973; and U.S. Pat. I want to be

"Human antibody" includes an antibody that has the amino acid sequence of a human immunoglobulin, and further includes an antibody isolated from a human immunoglobulin library, or that is transgenic with respect to one or more human immunoglobulins and that provides endogenous immunity. Contains antibodies isolated from animals that do not express globulins. Human antibodies can be made by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. U.S. Pat. No. 4,444,887 and U.S. Pat. No. 4,716,111, each of which is incorporated by reference in its entirety; and PCT International Publication No. WO 98/46645; International Publication No. 98/50433 pamphlet; International Publication No. 98/24893 pamphlet; International Publication No. 98/16654 pamphlet; International Publication No. 96/34096 pamphlet; International Publication No. 96/33735 pamphlet; and International Publication No. 91/ Please refer to pamphlet No. 10741. Human antibodies can also be produced by using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but are capable of expressing human immunoglobulin genes. For example, PCT WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735, all of which are incorporated herein by reference in their entirety. Pamphlet; U.S. Patent No. 5,413,923; No. 5,625,126; No. 5,633,425; No. 5,569,825; No. 5,661,016 No. 5,545,806; No. 5,814,318; No. 5,885,793; No. 5,916,771; and No. 5,939, See the '598 specification. Fully human antibodies that recognize selected epitopes can be generated using a technique called "guided selection." In this approach, a selected non-human monoclonal antibody, e.g. a mouse antibody, is used to guide the selection of fully human antibodies that recognize the same epitope (Jespers et al., 1988, Biotechnology 12:899-903). reference).

A "primatized antibody" includes monkey variable regions and human constant regions. Methods for producing primatized antibodies are known in the art. See, e.g., U.S. Pat. No. 5,658,570; U.S. Pat. No. 5,681,722; sea bream.

Anti-glycoMUC1 antibodies of the present disclosure include both full-length (intact) antibody molecules and antigen-binding fragments capable of binding glycoMUC1. Examples of antigen binding fragments include, by way of example and without limitation, Fab, Fab', F(ab') ₂ , Fv fragments, single chain Fv fragments and single domain fragments.

The Fab fragment contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1). Fab' fragments differ from Fab fragments by the addition of several residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. F(ab') fragments are produced by cleavage of disulfide bonds in the hinge cysteines of F(ab') ₂ pepsin digestion products. Additional chemical coupling of antibody fragments is known to those skilled in the art. Fab and F(ab') ₁ fragments lack the Fc fragment of intact antibodies, are cleared from the animal circulation more rapidly than intact antibodies, and may have less nonspecific tissue binding than intact antibodies (e.g., Wahl et al. et al., 1983, J. Nucl. Med. 24:316).

An "Fv" fragment is the smallest fragment of an antibody that contains a complete target recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight non-covalent association (V _H -V _L dimer). In this configuration, the three CDRs of each variable domain interact to define a target binding site on the surface of the V _H -V _L dimer. Often six CDRs confer target binding specificity to an antibody. However, in some cases, even a single variable domain (or half of an Fv containing only three target-specific CDRs) may recognize the target, albeit with lower affinity than the entire binding site. It may have the ability to bind to it.

A "single chain Fv" or "scFv" antigen-binding fragment comprises the V _H and V _L domains of an antibody, where these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the V _H and V _L domains, which allows the scFv to form the desired structure for target binding.

A "single domain antibody" is composed of a single V _H or V _L domain that exhibits sufficient affinity for glycoMUCl. In a specific embodiment, the single domain antibody is a camelized antibody (see, eg, Riechmann, 1999, Journal of Immunological Methods 231:25-38).

The anti-glycoMUC1 antibodies of the present disclosure may also be bispecific and other multispecific antibodies. Bispecific antibodies are monoclonal antibodies, often human or humanized antibodies, that have binding specificities for two different epitopes on the same or different antigens. In the present disclosure, one of the binding specificities may be directed to glycoMUC1 and the other to any other antigen, such as cell surface proteins, receptors, receptor subunits, tissue-specific antigens, It may be directed to proteins derived from viruses, envelope proteins encoded by viruses, proteins derived from bacteria, bacterial surface proteins, and the like. In certain preferred embodiments, bispecific and other multispecific anti-glyco-MUC1 antibodies and antigen-binding fragments are directed to a second MUC1 epitope, another protein that is coexpressed with MUC1 on cancer cells. or an epitope on another protein present on a different cell, such as an activated T cell. Bispecific antibodies of the present disclosure include IgG-based bispecific antibodies and single chain-based bispecific antibodies.

The IgG-mode bispecific antibodies of the present disclosure may be any of the various types of IgG-mode bispecific antibodies known in the art, such as quadroma bispecific antibodies, "knob-in-hole" knobs-in-hole)" bispecific antibody, CrossMab bispecific antibody, charge-paired bispecific antibody, general light chain bispecific antibody, one-arm single chain Fab - immunoglobulin Gamma Bispecific Antibodies, Disulfide Stabilized Fv Bispecific Antibodies, DuetMab, Controlled Fab Arm Exchange Bispecific Antibodies, Strand Exchange Engineered Domain Body Bispecific Antibodies, Two Arms leucine zipper heterodimeric monoclonal bispecific antibodies, κλ body bispecific antibodies, dual variable domain bispecific antibodies, and crossed dual variable domain bispecific antibodies. For example, Kohler and Milstein, 1975, Nature 256:495-497; Milstein and Cuello, 1983, Nature 305:537-40; Ridgway et al., 1996, Protein, which are incorporated herein by this reference in their entirety. Eng. 9:617-621; Schaefer et al., 2011, Proc Natl Acad Sci USA 108:11187-92; Gunasekaran et al., 2010, J Biol Chem 285:19637-46; Fischer et al., 2015 Nature Commun 6:6113; Schanzer et al., 2014, J Biol Chem 289:18693-706; Metz et al., 2012 Protein Eng Des Sel 25:571-80; Mazor et al., 2015 MAbs 7:377-89; Labrijn et al., 2013 Proc Natl Acad Sci USA 110:5145-50; Davis et al., 2010 Protein Eng Des Sel 23:195-202; Wranik et al., 2012, J Biol Chem 287:43331-9; Gu et al. al., 2015, PLoS One 10(5):e0124135; Steinmetz et al., 2016, MAbs 8(5):867-78; Klein et al., 2016, mAbs, 8(6):1010-1020; Liu See et al., 2017, Front. Immunol. 8:38; and Yang et al., 2017, Int. J. Mol. Sci. 18:48.

In some embodiments, the bispecific antibody of the present disclosure is a CrossMab. CrossMab technology is disclosed in WO 2009/080251, WO 2009/080252, WO 2009/080253, and WO 2009/2009, all of which are incorporated herein by this reference in their entirety. 080254 pamphlet, WO 2013/026833 pamphlet, WO 2016/020309 pamphlet, and Schaefer et al., 2011, Proc Natl Acad Sci USA 108:11187-92. Briefly, CrossMab technology is based on the crossing of domains between heavy and light chains within one Fab arm of a bispecific IgG that promotes correct chain association. The CrossMab bispecific antibodies of the present disclosure may be "CrossMab ^FAB " antibodies in which the heavy and light chains of the Fab portion of one arm of the bispecific IgG antibody are exchanged. In other embodiments, the CrossMab bispecific antibodies of the present disclosure are "CrossMab ^VH-VL" in which only the heavy and light chain variable domains of the Fab portion of one arm of the bispecific IgG antibody are exchanged. ” may be an antibody. In yet other embodiments, the CrossMab bispecific antibodies of the present disclosure are crossMab ^CH1- , in which only the heavy and light chain constant domains of the Fab portion of one arm of the bispecific IgG antibody are exchanged. ^CL '' antibody. The CrossMab ^CH1-CL antibody, in contrast to CrossMab ^FAB and CrossMab ^VH-VL , does not have the expected side products and therefore, in some embodiments, the CrossMab ^CH1-CL bispecific antibody is preferred. . See Klein et al., 2016, mAbs, 8(6):1010-1020. Further embodiments of the CrossMab of the present disclosure are described below in Section 5.2.

In some embodiments, the bispecific antibodies of the present disclosure are controlled Fab arm exchanged bispecific antibodies. Methods for making Fab arm-swapped bispecific antibodies are described in PCT Publication No. WO 2011/131746 and Labrijn et al., 2014 Nat Protoc. 9, which are incorporated herein by reference in their entirety. 10):2450-63. Briefly, regulated Fab arm-switched bispecific antibodies are produced by separately expressing two parental IgG1 containing a single matching point mutation in the CH3 domain, in vitro, under redox conditions. by mixing the parental IgG1 at the bottom to allow recombination of the half molecules and by removing the reductant and reoxidizing the interchain disulfide bonds, thereby forming a bispecific antibody. It can be made.

Bispecific antibodies of the present disclosure may include an Fc domain composed of a first and a second subunit. In one embodiment, the Fc domain is an IgG Fc domain. In certain embodiments, the Fc domain is _{an IgGi} Fc domain. In another embodiment, the Fc domain is an _IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG ₄ Fc domain comprising an amino acid substitution at position S228, particularly the amino acid substitution S228P (Kabat EU Index numbering). This amino acid substitution reduces Fab arm exchange of _IgG4 antibodies in vivo (see Stubenrauch et al., 2010, Drug Metabolism and Disposition 38:84-91). In further specific embodiments, the Fc domain is a human Fc domain. In an even more specific embodiment, the Fc domain is a human IgG ₁ Fc domain. An exemplary sequence of a human IgG ₁ Fc region is shown in SEQ ID NO:42.

In certain embodiments, the Fc domain includes a modification that promotes association of the first and second subunits of the Fc domain. The site of most extensive protein-protein interaction between the two subunits of the human IgG Fc domain is in the CH3 domain. Thus, in one embodiment said modification is in the CH3 domain of the Fc domain.

In a specific embodiment, the modification that promotes the association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, which It contains a "knob" modification in one of the two subunits of the domain and a "whole" modification in the other of the two subunits of the Fc domain. Knob-into-hole technology is described, for example, in US Pat. No. 5,731,168; US Pat. No. 7,695,936; Ridgway et al., 1996, Prot Eng 9:617-621, and Carter , J, 2001, Immunol Meth 248:7-15. Generally, the method includes adding protrusions to the boundaries of the first polypeptide such that the protrusions can enter the cavity so as to promote heterodimer formation and prevent homodimer formation. (a "knob"), and a corresponding cavity (a "hole") at the border of the second polypeptide. Protrusions are constructed by replacing small amino acid side chains from the border of the first polypeptide with larger side chains (eg, tyrosine or tryptophan). By replacing large amino acid side chains with smaller amino acid side chains (eg, alanine or threonine), a commensurate cavity is created at the border of the second polypeptide with the same or similar size as the protrusion.

Thus, in some embodiments, the CH3 domain of the first subunit of the Fc domain is replaced by replacing the amino acid residue in the CH3 domain of the first subunit with an amino acid residue that has a larger side chain volume. Within the domain, a protrusion is generated that can enter the cavity within the CH3 domain of the second subunit, allowing the amino acid residues in the CH3 domain of the second subunit of the Fc domain to be transferred to a smaller side chain volume. A cavity is created in the CH3 domain of the second subunit into which a protrusion in the CH3 domain of the first subunit can be placed. Preferably, said amino acid residue with a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, said amino acid residue with a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). Protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide, eg, by site-directed mutagenesis, or by peptide synthesis. An exemplary substitution is Y470T.

In certain such embodiments, in the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain, the threonine residue at position 407 is replaced with a tryptophan residue (T366W). The tyrosine residue is replaced with a valine residue (Y407V), the threonine residue at position 366 is optionally replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue. (L368A) (numbering according to Kabat EU index). In a further embodiment, in the first subunit of the Fc domain, in addition, the serine residue at position 354 is replaced with a cysteine residue (S354C), or the glutamic acid residue at position 356 is replaced with a cysteine residue. (E356C) (in particular, the serine residue at position 354 is replaced by a cysteine residue), and in addition, the tyrosine residue at position 349 is replaced by a cysteine residue in the second subunit of the Fc domain. (Y349C) (numbering according to Kabat EU index). In certain embodiments, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (per Kabat EU Index). numbering).

In some embodiments, fixed steering (e.g., as described in Gunasekaran et al., 2010, J Biol Chem 285(25):19637-46) is used to control the first and second positions of the Fc domain. can promote the association of subunits.

In some embodiments, the Fc domain includes one or more amino acid substitutions that reduce binding to Fc receptors and/or effector functions.

In certain embodiments, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is selected from the group of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and cytokine secretion. One or more. In certain embodiments, the effector function is ADCC.

Typically, the same amino acid substitution or substitutions are present in each of the two subunits of the Fc domain. In one embodiment, one or more amino acid substitutions reduce the binding affinity of the Fc domain to an Fc receptor. In one embodiment, the one or more amino acid substitutions reduce the binding affinity of the Fc domain to an Fc receptor by at least a factor of 2, at least by a factor of 5, or by a factor of at least 10.

In one embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group E233, L234, L235, N297, P331 and P329 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group L234, L235 and P329 (numbering according to the Kabat EU index). In some embodiments, the Fc domain comprises amino acid substitutions L234A and L235A (numbering according to Kabat EU index). In one such embodiment, the Fc domain is an IgG ₁ Fc domain, particularly a human IgG ₁ Fc domain. In one embodiment, the Fc domain includes an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, especially P329G (numbering according to the Kabat EU index). In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and an additional amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numbering according to the Kabat EU index). In more specific embodiments, the additional amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In certain embodiments, the Fc domain includes amino acid substitutions at positions P329, L234 and L235 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises amino acid mutations L234A, L235A and P329G ("P329G LALA", "PGLALA" or "LALAPG"). Specifically, in certain embodiments, each subunit of the Fc domain comprises amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering), i.e., the first and second subunits of the Fc domain In each of the following, the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A), and the proline residue at position 329 is replaced by a glycine residue. (P329G) (numbering according to Kabat EU index). In one such embodiment, the Fc domain is an IgG ₁ Fc domain, particularly a human IgG ₁ Fc domain.

The single chain-based bispecific antibodies of the present disclosure are compatible with various types of single chain-based bispecific antibodies known in the art, such as bispecific T cell derivatives (BiTEs) (bispecific T cell derivatives). (BiTE antibodies), diabodies, tandem diabodies (tandabs), dual-affinity retargeting molecules (DARTs), and bispecific killer cell derivatives. It can be. For example, Loffler et al., 2000, Blood 95:2098-103; Holliger et al., 1993, Proc Natl Acad Sci USA, 90:6444-8; Kipriyanov et al., herein incorporated by reference in their entirety. al., 1999, Mol Biol 293:41-56; Johnson et al., 2010, Mol Biol 399:436-49; Wiernik et al., 2013, Clin Cancer Res 19:3844-55; Liu et al., 2017 , Front. Immunol. 8:38; and Yang et al., 2017, Int. J. Mol. Sci. 18:48.

In some embodiments, the bispecific antibodies of the present disclosure are bispecific T cell derivatives (BiTEs). BiTEs are single polypeptide chain molecules with two antigen-binding domains, one of which binds to a T-cell antigen and the second antigen-binding domain binds to an antigen presented on the surface of a target. (PCT Publication No. WO 05/061547, herein incorporated by reference in its entirety; Baeuerle et al., 2008, Drugs of the Future 33: 137-147; Bargou, et al., 2008 , Science 321:974-977). Accordingly, the BiTEs of the present disclosure have an antigen binding domain that binds a T cell antigen and a second antigen binding domain directed to glycoMUC1.

In some embodiments, a bispecific antibody of the present disclosure is a dual affinity retargeting molecule (DART). DART comprises at least two polypeptide chains that associate (particularly through covalent interactions) to form at least two epitope binding sites, which may recognize the same epitope. or may recognize different epitopes. Each of the polypeptide chains of DART includes an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region, but these regions do not interact to form an epitope binding site. Rather, the immunoglobulin heavy chain variable region of one (e.g., first) DART polypeptide chain is compatible with the immunoglobulin light chain variable region of a different (e.g., second) DART™ polypeptide chain. act to form an epitope binding site. Similarly, the immunoglobulin light chain variable region of one (e.g., first) DART polypeptide chain interacts with a different (e.g., second) immunoglobulin heavy chain variable region of the DART polypeptide chain. , forming an epitope binding site. DARTs may be monospecific, bispecific, trispecific, etc., and thus contain one, two, three or more different epitopes (these may be of the same or different antigens). ) can be combined at the same time. DART may additionally be monovalent, divalent, trivalent, tetravalent, pentavalent, hexavalent, etc., and thus contain 1, 2, 3, 4, 5, 6 or more molecules. Can be combined at the same time. These two properties of DART (i.e., degree of specificity and valence) combine to create, for example, a quadrivalent (i.e., capable of binding four sets of epitopes) bispecific antibodies (i.e., two PCT WO 2006/113665; It is disclosed in pamphlet No. 157379 and pamphlet WO 2010/080538.

In some embodiments of the bispecific antibodies of the present disclosure, one of the binding specificities is directed against glycoMUCl and the other is directed against an antigen expressed on immune effector cells. The term "immune effector cell" or "effector cell" as used herein refers to a member of the natural repertoire of cells in the mammalian immune system that can be activated to influence the viability of target cells. Refers to cells. Immune effector cells include lymphoid cells, such as natural killer (NK) cells, T cells, including cytotoxic T cells, or B cells, as well as monocytes or macrophages, dendritic cells, and Myeloid cells such as neutrophil granulocytes can also be considered immune effector cells. Accordingly, said effector cells are preferably NK cells, T cells, B cells, monocytes, macrophages, dendritic cells or neutrophil granulocytes. Recruitment of effector cells to abnormal cells is such that immune effector cells can directly kill or indirectly initiate the killing of the abnormal cells to which they are recruited. This means that it is taken up in close proximity to target cells. To avoid non-specific interactions, the bispecific antibodies of the present disclosure target antigens on immune effector cells that are at least overexpressed by these immune effector cells compared to other cells in the body. It is preferable to recognize specifically. Target antigens presented on immune effector cells can include CD3, CD8, CD16, CD25, CD28, CD64, CD89, NKG2D and NKp46. Preferably, the antigen on the immune effector cells is CD3 expressed on T cells.

As used herein, "CD3" refers to mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. refers to any naturally occurring CD3 from any vertebrate source, including. The term encompasses "full length" unprocessed CD3, as well as all forms of CD3 that result from processing in the cell. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. The most preferred antigen on immune effector cells is the CD3 epsilon chain. This antigen has been shown to be highly effective in recruiting T cells to abnormal cells. Therefore, the bispecific antibodies of the present disclosure preferably specifically recognize CD3 epsilon. The amino acid sequence of human CD3 epsilon is shown in UniProt (www.uniprot.org) accession number P07766 (version 144) or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_000724.1. The amino acid sequence of cynomolgus monkey [Macaca fascicularis] CD3 epsilon is shown in NCBI GenBank number BAB71849.1. For human therapeutic use, bispecific antibodies are used whose CD3 binding domain specifically binds human CD3 (eg, human CD3 epsilon chain). For preclinical studies in non-human animals or cell lines, bispecificity in which the CD3-binding domain specifically binds to CD3 in the species used for preclinical studies (e.g., cynomolgus monkey CD3 for primate studies) Antibodies can be used.

As used herein, a binding domain that "specifically binds" or "specifically recognizes" a target antigen from a particular species refers to a binding domain that "specifically binds to" or "specifically recognizes" a target antigen from a particular species. and therefore encompass antibodies in which one or more of the binding domains has cross-species cross-reactivity. For example, a CD3 binding domain that "specifically binds to" or "specifically recognizes" human CD3 can also bind to or recognize cyomolgus monkey CD3, and vice versa. It is.

In some embodiments, the bispecific antibodies of the present disclosure can compete with monoclonal antibody H2C (described in PCT Publication No. WO 2008/119567) for binding to an epitope of CD3. In other embodiments, the bispecific antibodies of the present disclosure are monoclonal antibody V9 (Rodrigues et al., 1992, Int J Cancer Suppl 7:45-50 and U.S. Pat. No. 054,297). In yet other embodiments, the bispecific antibodies of the present disclosure combine monoclonal antibody FN18 (described in Nooij et al., 1986, Eur J Immunol 19:981-984) with respect to binding to an epitope of CD3. can compete. In yet other embodiments, the bispecific antibodies of the present disclosure compete with monoclonal antibody SP34 (described in Pessano et al., 1985, EMBO J 4:337-340) for binding to an epitope of CD3. can do.

Anti-glycoMUC1 antibodies of the present disclosure include derivatized antibodies. For example, but not limited to, derivatized antibodies typically include glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, cellular Modified by linkage to a ligand or other protein. Any of the various chemical modifications can be performed by known techniques such as, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin. In addition, the derivatives may contain one or more unnatural amino acids, e.g. using ambrx technology (see e.g. Wolfson, 2006, Chem. Biol. 13(10):1011-2). reference).

An anti-glycoMUC1 antibody or binding fragment can be an antibody or fragment whose sequence has been modified to alter the biological effector function mediated by at least one constant region. For example, in some embodiments, an anti-glycoMUC1 antibody may be modified to reduce at least one constant region-mediated biological effector function relative to an unmodified antibody, e.g. , modified to reduce binding to Fc receptors (FcγR). Binding to FcγRs can be reduced by mutating the immunoglobulin constant region segments of antibodies in specific regions required for FcγR interaction (e.g., Canfield and Morrison, 1991, J. Exp. Med. 173 :1483-1491; and Lund et al., 1991, J. Immunol. 147:2657-2662). Reducing the FcγR binding ability of antibodies can also reduce other effector functions that rely on FcγR interactions, such as opsonization, phagocytosis, and antigen-dependent cellular cytotoxicity (“ADCC”).

The anti-glycoMUC1 antibodies or binding fragments described herein acquire or improve at least one constant region-mediated biological effector function relative to unmodified antibodies, e.g., FcγR including antibodies and/or binding fragments that have been modified to enhance interaction (see, eg, US Patent Application Publication No. 2006/0134709). For example, an anti-glycoMUC1 antibody of the present disclosure may have a constant region that binds FcγRIIA, FcγRIIB, and/or FcγRIIIA with greater affinity than the corresponding wild-type constant region.

Thus, antibodies of the present disclosure may have alterations in biological activity that cause an increase or decrease in opsonization, phagocytosis, or ADCC. Such modifications are known in the art. For example, modifications in antibodies that reduce ADCC activity are described in US Pat. No. 5,834,597. An exemplary ADCC-reducing variant corresponds to “mutant 3” in which residue 236 is deleted and residues 234, 235 and 237 (using EU numbering) are replaced with alanine (US 4 of the '597 patent).

In some embodiments, the anti-glycoMUC1 antibodies of the present disclosure have low levels of fucose or lack fucose. Antibodies lacking fucose have been shown to correlate with enhanced ADCC activity, especially at low doses of antibody. See Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278:3466-73. Methods for preparing low-fucose antibodies include growth in rat myeloma YB2/0 cells (ATCC CRL 1662). YB2/0 cells express low levels of FUT8 mRNA, which encodes α-1,6-fucosyltransferase, an enzyme required for polypeptide fucosylation.

In yet another embodiment, an anti-glycoMUC1 antibody or binding fragment improves its binding affinity to the fetal Fc receptor, FcRn, for example, by mutating immunoglobulin constant region segments in specific regions involved in FcRn interaction. (See, for example, WO 2005/123780 pamphlet). In certain embodiments, the IgG class anti-glycoMUC1 antibody has at least one of amino acid residues 250, 314, and 428 of the heavy chain constant region, such as positions 250 and 428, or positions 250 and 314, or 314 and 428. or positions 250, 314, and 428, alone or in any combination thereof; a specific combination is positions 250 and 428. In the case of position 250, the substituted amino acid residue may be any amino acid residue other than threonine, including, but not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, It may be isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan, or tyrosine. In the case of position 314, the substituted amino acid residue may be any amino acid residue other than leucine, including, but not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, It may be isoleucine, lysine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine. In the case of position 428, the amino acid residue substituted may be any amino acid residue other than methionine, including, but not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, It may be isoleucine, lysine, leucine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine. Specific combinations of suitable amino acid substitutions are identified in Table 1 of US Pat. No. 7,217,797, which is incorporated herein by reference. Such mutations increase binding to FcRn, thereby protecting the antibody from degradation and increasing its half-life.

In yet other embodiments, the anti-glycoMUC1 antibodies of the presently disclosed antigen-binding fragments are as described, for example, in Jung and Pluckthun, 1997, Protein Engineering 10:9, 959-966; Yazaki et al., 2004, Protein Eng. Des Sel. 17 (5):481-9. Epub 2004 Aug. 17; Contains amino acids.

In yet other embodiments particularly useful for diagnostic applications, the anti-glycoMUC1 antibodies of the antigen-binding fragments of the present disclosure are attached to a detectable moiety. Detectable moieties include radioactive moieties, colorimetric molecules, fluorescent moieties, chemiluminescent moieties, antigens, enzymes, detectable beads (e.g. magnetic or high electron density (e.g. gold) beads), or conjugated to another molecule. (eg, biotin or streptavidin).

Radioisotopes or radionuclides can include ^3H , ^14C , ^15N , ^35S , ^90Y , ^99Tc , ^111In , ^125I , ^131I .

Fluorescent labels include rhodamine, lanthanide fluorescence, fluorescein and its derivatives, fluorescent dyes, GFP (GFP stands for "Green Fluorescent Protein"), dansyl, umbelliferone, phycoerythrin, phycocyanin, allophycocyanin, o - phthaldehyde, and fluorescamine may be mentioned.

Enzyme labels include horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose-6-phosphate dehydrogenase (“G6PDH”), alpha-D-galactosidase, glucose oxidase, glucose amylase, carbonic anhydrase, acetylcholinesterase, and lysozyme. , malate dehydrogenase and peroxidase.

Chemiluminescent labels or chemiluminescers, such as isoluminol, luminol and dioxetane

Other detectable moieties include molecules such as biotin, digoxigenin or 5-bromodeoxyuridine.

In certain embodiments, an anti-glycoMUC1 antibody or antigen-binding fragment of the present disclosure competes with an antibody or antigen-binding fragment comprising GO2 or the heavy and light chain variable regions of GO2 (SEQ ID NO: 3 and 4, respectively).

Competition is achieved with cells expressing glyco-MUC1 epitopes that bind GO2 or with glycosylated MUC1 peptides containing epitopes that bind GO2, such as GalNAc- The 60-mer peptide glycosylated with T2, GalNAc-T4, and GalNAc-T1 (VTSAPDTRPAPGSTAPPAHG) ₃ can be assayed. As a control, cells that do not express the epitope or non-glycosylated peptides can be used.

Cells on which competition assays can be performed include, but are not limited to, breast cancer cell lines MCF7 or T47D, and recombinant cells engineered to express the glycoMUC1 epitope. In one non-limiting example, CHO IdID cells lack UDP-Gal/GalNAc epimerase and have insufficient O-glycosylation and galactosylation of GalNAc in the absence of exogenous addition of GalNAc and Gal, respectively. are engineered to express MUC1 and grow in the absence or presence of GalNAc, resulting in cells expressing the Tn glycoform of MUC1 to which GO2 is bound in the latter case. Cells expressing an unglycosylated form of MUC1 can be used as a negative control.

Assays for competition include, but are not limited to, radioactively labeled immunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), sandwich ELISAs, fluorescence-activated cell sorting (FACS) assays, and Biacore assays. .

In performing an antibody competition assay (regardless of species or isotype) between a reference antibody and a test antibody, a detectable label, e.g. fluorophore, biotin or The reference may be labeled with an enzyme label (or even a radioactive label). In this case, cells expressing glycoMUC1 are incubated with unlabeled test antibody, labeled reference antibody is added, and the intensity of bound label is measured. If the test antibody competes with the labeled reference antibody by binding to overlapping epitopes, the intensity is expected to decrease compared to control reactions performed without the test antibody.

In a specific embodiment of this assay, the concentration of labeled reference antibody that results in 80% of maximal binding under assay conditions (e.g., specified cell density) ("conc _80% ") is first determined; Competition assays are performed at 10× conc _80% of unlabeled test antibody and conc _80% of labeled reference antibody.

Inhibition can be expressed as the inhibition constant, or K _i , which has the following formula:
K _i =IC ₅₀ /(1+[reference Ab concentration]/K _d )
where the IC ₅₀ is the concentration of the test antibody that results in a 50% reduction in the binding of the reference antibody, and the K _d is the dissociation constant of the reference antibody, which is the It is a measure. Antibodies that compete with the anti-glycoMUC1 antibodies disclosed herein can have a K _i of 10 pM to 10 nM under the assay conditions described herein.

In various embodiments, at a reference antibody concentration that is 80% of maximal binding under the particular assay conditions used, and a test antibody concentration that is 10 times higher than the reference antibody concentration, at least about 20% or more, e.g. , at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, or a percentage ranging between any of the foregoing values; A test antibody is considered to compete with a reference antibody if the test antibody decreases the binding of the reference antibody.

In one example of a competition assay, a 60-mer peptide that is glycosylated MUC1 is transferred to a solid surface, e.g., by contacting the plate with a solution of the peptide (e.g., overnight at 4°C at a concentration of 1 μg/mL in PBS). Adhere on microwell plate. Plates are washed (eg, 0.1% Tween 20 in PBS) and blocked (eg, in Superblock, Thermo Scientific, Rockford, IL). Subsaturating amounts of biotinylated GO2 (e.g., at a concentration of 80 ng/mL) in ELISA buffer (e.g., 1% BSA and 0.1% Tween 20 in PBS) and serial dilutions (e.g., 2. Add a mixture of unlabeled GO2 (“reference” antibody) or competing anti-glycoMUC1 antibody (“test” antibody) antibodies to the wells at a concentration of 8 μg/mL, 8.3 μg/mL, or 25 μg/mL and incubate the plate for 1 hour with gentle shaking. Wash the plate, add 1 μg/mL HRP-conjugated streptavidin diluted in ELISA buffer to each well, and incubate the plate for 1 hour. Plates were washed and bound antibodies were detected by addition of substrate (eg, TMB, Biofx Laboratories Inc., Owings Mills, MD). Stopped buffer (eg, Bioo FX STOP reagent, BiOFX LABORATORIES Inc., Owings Mills, MD) stopped the reaction, and stopped the reaction and microplate readers (eg, Molecular Devi. Use CES, SunnyVale, CA) Measure at 650 nm.

Variations on this competition assay can also be used to test competition between GO2 and another anti-glycoMUC1 antibody. For example, in certain embodiments, anti-glycoMUC1 antibody is used as a reference antibody and GO2 is used as a test antibody. Additionally, instead of a 60-mer peptide that is glycosylated MUC1, membrane-bound glyco-MUC1 expressed on the cell surface during culture (e.g. on the surface of one of the cell types mentioned above) may be used. good. Generally, about 10 ⁴ to 10 ⁶ transformants are used, such as about 10 ⁵ transformants. Other formats of competition assays are known in the art and can be employed.

In various embodiments, the anti-glycoMUC1 antibodies of the present disclosure have a concentration of 0.08 μg/mL, 0.4 μg/mL, 2 μg/mL, 10 μg/mL, 50 μg/mL, 100 μg/mL. or at concentrations ranging between any of the aforementioned values (e.g., at concentrations ranging from 2 μg/mL to 10 μg/mL), the binding of labeled GO2 is reduced by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or a percentage range between any of the foregoing values (e.g., the anti-glyco-MUC1 antibodies of the present disclosure may be labeled (reducing GO2 binding by 50% to 70%).

In other embodiments, GO2 is present at a concentration of 0.4 μg/mL, 2 μg/mL, 10 μg/mL, 50 μg/mL, 250 μg/mL, or at a concentration range between any of the foregoing values (e.g. , 2 μg/mL to 10 μg/mL), GO2 inhibits the binding of the labeled anti-glyco-MUC1 antibodies of the present disclosure by at least 40%, at least 50%, at least 60%, at least 70%. %, at least 80%, at least 90%, or any of the foregoing values (e.g., GO2 reduces the binding of the labeled anti-glycoMUC1 antibodies of the present disclosure by 50% to 70%). % reduction).

In the aforementioned assays, the GO2 antibody can be replaced with any antibody or antigen-binding fragment that includes the CDRs or heavy and light chain variable regions of GO2, such as a humanized or chimeric counterpart of GO2.

In certain embodiments, the anti-glycoMUC1 antibodies or antigen-binding fragments of the present disclosure comprise (or are encoded by) the heavy chain and/or light chain variable sequences set forth in Table 1. In other embodiments, the anti-glycoMUCl antibodies or antigen-binding fragments of the present disclosure comprise (or are encoded by the nucleotide sequences) the heavy chain and/or light chain CDR sequences set forth in Table 1. The framework sequences of such anti-glyco-MUC1 antibodies and antigen-binding fragments may be the native murine framework sequences listed in Table 1, or may be non-natural (e.g., humanized or human) framework sequences. It's okay.

In yet other embodiments, the present disclosure provides an anti-MUC1 antibody having heavy and light chain variable regions having at least 95%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO: 3 and 4, respectively. or provide an antigen-binding fragment.

In yet other embodiments, the anti-glycoMUC1 antibodies or antigen binding fragments of the present disclosure are single chain variable fragments (scFv). An exemplary scFv includes a heavy chain variable fragment N-terminal to a light chain variable fragment. In some embodiments, the heavy and light chain variable fragments of the scFv are covalently linked to a linker sequence of 4-15 amino acids. The scFv may be in the form of a bispecific T cell derivative or within a chimeric antigen receptor (CAR).

5.2 Anti-GlycoMUC1 and Anti-CD3 Bispecific Antibodies In some embodiments, the bispecific antibodies of the present disclosure are specific for CD3 (e.g., those comprising the CDRs or VH and VL listed in Table 4). and a second antigen-binding domain that specifically binds to glycoMUC1. The second antigen-binding domain may include the features described above for the glycoMUC1 antibody, alone or in combination (e.g., a combination of CDRs identified in Tables 1-3, e.g. or the VH and VL sequences identified in Table 1).

In some embodiments, the first antigen binding domain comprises a heavy chain variable region comprising a heavy chain CDR-H1 of SEQ ID NO: 34, a CDR-H2 of SEQ ID NO: 35, and a CDR-H3 of SEQ ID NO: 36; It comprises a light chain variable region comprising light chain CDR-L1 of number 37, CDR-L2 of SEQ ID NO: 38, and CDR-L3 of SEQ ID NO: 39.

In some embodiments, the second antigen binding domain comprises a CDR comprising the amino acid sequence of any of the CDR combinations set forth in numbered embodiments 3 to 17, e.g., (i) the heavy chain of SEQ ID NO: 5. A heavy chain variable region comprising CDR-H1, CDR-H2 of SEQ ID NO: 6, and CDR-H3 of SEQ ID NO: 7; and light chain CDR (CDR-L) 1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9, and It contains a light chain variable region including CDR-L3 of SEQ ID NO: 10.

In certain embodiments, the bispecific antibody is
(i) A heavy protein that specifically binds to CD3 and includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 34, CDR-H2 containing the amino acid sequence of SEQ ID NO: 35, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 36. and a light chain variable region comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 39. a first antigen-binding domain; and (ii) a CDR-H1 that specifically binds to glycoMUC1 and (i) comprises an amino acid sequence of SEQ ID NO: 33, more preferably a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 5; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, more preferably CDR-H1 comprising the amino acid sequence of SEQ ID NO: 6, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25, more preferably the amino acid sequence of SEQ ID NO: 7. heavy chain variable region comprising CDR-H3 comprising; and CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9 and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 31, more preferably comprises a second antigen binding domain comprising a light chain variable region comprising a CDR-L3 comprising the amino acid sequence of SEQ ID NO:10.

In some embodiments, the first antigen binding domain has a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 40, and A light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 41.

In some embodiments, the first antigen binding domain comprises the heavy chain variable region sequence of SEQ ID NO: 40 and the light chain variable region sequence of SEQ ID NO: 41.

In some embodiments, the second antigen binding domain has a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3, and A light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:4.

In some embodiments, the second antigen binding domain comprises the heavy chain variable region sequence of SEQ ID NO: 3 and the light chain variable region sequence of SEQ ID NO: 4.

In some embodiments, the first and/or second antigen binding domain is a Fab molecule. In some embodiments, the first antigen binding domain is a crossed Fab molecule in which either the variable or constant regions of the Fab light chain and Fab heavy chain are exchanged. In such embodiments, the second antigen binding domain is preferably a conventional Fab molecule.

In some embodiments, the first and second antigen-binding domains of the bispecific antibody are both Fab molecules, and in one of the antigen-binding domains (particularly the first antigen-binding domain) the Fab light chain and the variable domains VL and VH of the Fab heavy chain are replaced with each other,
i) In the constant domain CL of the first antigen-binding domain, the amino acid at position 124 is replaced with a positively charged amino acid (numbering according to Kabat) and in the constant domain CH1 of the first antigen-binding domain, the amino acid at position 147 or ii) in the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is substituted with a negatively charged amino acid (numbering according to the Kabat EU index); an amino acid with a positive charge (numbering according to Kabat), and in the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is replaced with an amino acid with a negative charge (Numbering according to Kabat EU index).

A bispecific antibody does not contain both the modifications mentioned in i) and ii). The constant domains CL and CH1 of the antigen binding domain with VH/VL exchange are not replaced with each other (ie, they remain unexchanged).

In a more specific embodiment,
i) In the constant domain CL of the first antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat); In the constant domain CH1 of the antigen-binding domain of No. 1, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index); or ii) in the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat); In the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index) .

In one such embodiment, in the constant domain CL of the second antigen binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H). (numbering according to Kabat), in the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E), or aspartic acid (D) ( Kabat EU index numbering).

In a further embodiment, in the constant domain CL of the second antigen binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat). ), in the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

In certain embodiments, in the constant domain CL of the second antigen binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat). ), the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) in the constant domain CH1 of the second antigen-binding domain. The amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index), and the amino acid at position 213 is replaced with glutamic acid (E) or aspartic acid (D). ) (numbering according to the Kabat EU index).

In a more specific embodiment, in the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is replaced with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is replaced with lysine (K). ) (numbering according to Kabat), and in the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 is replaced with glutamic acid (E) (numbering according to Kabat EU index), 213 The amino acid at position is substituted with glutamic acid (E) (numbering according to Kabat EU index).

In an even more specific embodiment, in the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is replaced with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is replaced with arginine ( R) (numbering according to Kabat), and in the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to Kabat EU index), The amino acid at position 213 is substituted with glutamic acid (E) (numbering according to Kabat EU index).

In certain embodiments, when the amino acid substitutions according to the above embodiments are made in constant domain CL and constant domain CH1 of the second antigen-binding domain, the constant domain CL of the second antigen-binding domain is a constant of the kappa isotype. Domain CL.

In some embodiments, the first and second antigen binding domains are fused to each other, optionally via a peptide linker.

In some embodiments, the first and second antigen-binding domains are each Fab molecules, and (i) the second antigen-binding domain is at the C-terminus of the Fab heavy chain and of the first antigen-binding domain. (ii) the first antigen-binding domain is C-terminus of the Fab heavy chain and the second antigen-binding domain is fused to the N-terminus of the Fab heavy chain; Either.

In some embodiments, bispecific antibodies provide monovalent binding to CD3.

In certain embodiments, the bispecific antibody comprises a single antigen binding domain that specifically binds CD3 and two antigen binding domains that specifically binds glycoMUC1. Thus, in some embodiments, the bispecific antibody comprises a third antigen binding domain that specifically binds glycoMUC1. In some embodiments, the third antigen moiety is identical to the first antigen binding domain (eg, is also a Fab molecule and includes the same amino acid sequence).

In certain embodiments, the bispecific antibody further comprises an Fc domain composed of a first and a second subunit. In one embodiment, the Fc domain is an IgG Fc domain. In certain embodiments, the Fc domain is _{an IgGi} Fc domain. In another embodiment, the Fc domain is an _IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG ₄ Fc domain comprising an amino acid substitution at position S228, in particular the amino acid substitution S228P (Kabat EU Index numbering). In further specific embodiments, the Fc domain is a human Fc domain. In an even more specific embodiment, the Fc domain is a human IgG ₁ Fc domain. An exemplary sequence of a human IgG ₁ Fc region is shown in SEQ ID NO:42.

In some embodiments, the first, second, and, if present, third antigen-binding domain are each a Fab molecule, and (a)(i) the second antigen-binding domain is of a Fab heavy chain. fused at the C-terminus to the N-terminus of the Fab heavy chain of a first antigen-binding domain; or (ii) the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain and the second antigen-binding domain is fused to the N-terminus of the Fab heavy chain; the domain is either fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain; (b) the third antigen-binding domain, if present, is fused to the Fab heavy chain; At the C-terminus of the chain, it is fused to the N-terminus of the second subunit of the Fc domain.

In certain embodiments, the Fc domain includes a modification that promotes association of the first and second subunits of the Fc domain, eg, as described in Section 5.1.

In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and/or effector functions, eg, as described in Section 5.1.

In certain embodiments, the bispecific antibody is
(i) a first antigen-binding domain that specifically binds to CD3, the first antigen-binding domain being a crossed Fab molecule and comprising either the variable or constant region of a Fab light chain and a Fab heavy chain; in particular a first antigen-binding domain, in which the variable region has been exchanged;
(ii) second and third antigen-binding domains that specifically bind to glycoMUC1, which are heavy chain CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6, and CDR-H3 of SEQ ID NO: 7; and a light chain variable region comprising a light chain CDR-L1 of SEQ ID NO: 8, a CDR-L2 of SEQ ID NO: 9, and a CDR-L3 of SEQ ID NO: 10; a second and third antigen-binding domain, each of which is a Fab molecule, in particular a conventional Fab molecule;
(iii) comprising an Fc domain composed of a first and second subunit capable of stable association;
the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain; , fused to the N-terminus of the first subunit of the Fc domain, and the third antigen-binding domain is fused to the N-terminus of the second subunit of the Fc domain, at the C-terminus of the Fab heavy chain. .

In one embodiment, the first antigen binding domain is a heavy chain variable region comprising heavy chain CDR-H1 of SEQ ID NO: 34, CDR-H2 of SEQ ID NO: 35, and CDR-H3 of SEQ ID NO: 36; and SEQ ID NO: 37. The light chain variable region comprises the light chain CDR-L1 of SEQ ID NO: 38, the CDR-L2 of SEQ ID NO: 38, and the CDR-L3 of SEQ ID NO: 39.

In one embodiment, the first antigen binding domain has a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 40; A light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to a 41 amino acid sequence.

In one embodiment, the first antigen binding domain comprises the heavy chain variable region sequence of SEQ ID NO: 40 and the light chain variable region sequence of SEQ ID NO: 41.

In one embodiment, the second and third antigen binding domains have heavy chain variable region sequences that are at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3; and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:4. Preferably, the antigen binding domain comprises CDRs comprising the amino acid sequence of any of the CDR combinations set out in numbered embodiments 3 to 17. In one embodiment, the second and third antigen binding domains comprise the heavy chain variable region of SEQ ID NO: 3 and the light chain variable region of SEQ ID NO: 4.

Fc domains according to the above embodiments may include all of the features described above for Fc domains, alone or in combination.

In some embodiments, the antigen binding domain and the Fc region are fused together by a peptide linker, such as in SEQ ID NO: 45 and SEQ ID NO: 46.

In one embodiment, in the constant domain CL of the second and third Fab molecules of (ii), the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is , lysine (K) or arginine (R), in particular arginine (R) (numbering according to Kabat), in the constant domain CH1 of the second and third Fab molecules of (ii) at position 147. The amino acid is substituted with glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to Kabat EU index).

In one embodiment, the bispecific antibody is a polypeptide comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 43. (preferably includes CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 31), and the sequence of SEQ ID NO: 44. A polypeptide comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical sequences (preferably CD3 heavy chain and light chain CDR sequences listed in Table 4). ), a polypeptide comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 45 (preferably the amino acid sequence of SEQ ID NO: 33); at least 80%, 85%, 90% with the sequence SEQ ID NO: 46); , 95%, 96%, 97%, 98%, or 99% identical sequences (preferably CDR-H1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29) , CDR-H3 containing the amino acid sequence of SEQ ID NO: 25, CDR-L1 containing the amino acid sequence of SEQ ID NO: 37, CDR-L2 containing the amino acid sequence of SEQ ID NO: 38, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 39. including).

In one embodiment, the bispecific antibody comprises a polypeptide (particularly two polypeptides) comprising the sequence SEQ ID NO: 43, a polypeptide comprising the sequence SEQ ID NO: 44, a polypeptide comprising the sequence SEQ ID NO: 45, and A polypeptide comprising the sequence of SEQ ID NO:46.

5.3 Antibody-Drug Conjugates Another aspect of the present disclosure relates to antibody drug conjugates (ADCs) comprising the anti-glycoMUC1 antibodies and antigen binding fragments of the present disclosure. ADC generally comprises an anti-glycoMUC1 antibody as described herein to which one or more cytotoxic and/or cytostatic substances are linked via one or more linkers. and/or binding fragments. In specific embodiments, the ADC has structural formula (I):
[DL-XY] _n -Ab
or a salt thereof, in which each "D" independently represents a cytotoxic substance and/or a cytostatic substance (a "drug");"Ab" represents an anti-glycoMUC1 antigen binding domain, such as an anti-glycoMUC1 antibody or binding fragment described herein; each "XY" represents a functional group R ^x on the linker; and a "complementary" functional group R ^y on the antibody, and n represents the number of drugs linked to the ADC, or the drug-to-antibody ratio (DAR) of the ADC.

Specific embodiments of various antibodies (Abs) that may include ADCs include the various embodiments of anti-glycoMUC1 antibodies and/or binding fragments described above.

In some specific embodiments of ADCs and/or salts of structural formula (I), each D is the same and/or each L is the same.

In addition to the cytotoxic and/or cytostatic substances (D) and linkers (L) that may constitute the anti-glyco-MUC1 ADCs of the present disclosure, there are a number of cytotoxic substances and/or cells linked to the ADCs. Specific embodiments of antiproliferative substances are described in more detail below.

5.3.1. Cytotoxic and/or cytostatic substances Cytotoxic and/or cytostatic substances inhibit the proliferation and/or replication of cells, in particular cancer and/or tumor cells, and/or It can be any drug known to kill such cells. A large number of drugs with cytotoxic and/or cytostatic properties are known in the literature. Non-limiting examples of classes of cytotoxic and/or cytostatic agents include, by way of example and without limitation, radionuclides, alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA Intercalating agents (e.g., minor groove binders), RNA/DNA antimetabolites, cell cycle modulators, kinase inhibitors, protein synthesis inhibitors, histone deacetylase inhibitors, mitochondrial inhibitors, Examples include mitotic agents.

Specific non-limiting examples of agents within certain of these various classes are provided below.

Alkylating agent: asaley ((L-leucine, N-[N-acetyl-4-[bis-(2-chloroethyl)amino]-DL-phenylalanyl]-, ethyl ester; NSC167780; CAS registration number 3577897)); AZQ ((1,4-cyclohexadiene-1,4-dicarbamic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo-, diethyl ester; NSC182986; CAS registration number 57998682)) ; BCNU ((N,N'-bis(2-chloroethyl)-N-nitrosourea; NSC409962; CAS registration number 154938)); Busulfan (1,4-butanediol dimethane sulfonate; NSC750; CAS registration number 55981); (carboxyphthalato)platinum (NSC27164; CAS registration number 65296813); CBDCA ((cis-(1,1-cyclobutanedicarboxylate)diamineplatinum(II)); NSC241240; CAS registration number 41575944)); CCNU ((N -(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea; NSC79037; CAS registration number 13010474)); CHIP (iproplatin; NSC256927); chlorambucil (NSC3088; CAS registration number 305033); chlorozotocin ((2-[[ [(2-chloroethyl)nitrosamino]carbonyl]amino]-2-deoxy-D-glucopyranose; NSC178248; CAS Registry No. 54749905)); Cisplatin (cisplatin; NSC119875; CAS Registry No. 15663271); Clomesone (NSC338947) ; CAS registration number 88343720); cyanomorpholinodoxorubicin (NCS357704; CAS registration number 88254073); cyclodisone (NSC348948; CAS registration number 99591738); dianhydrogalactitol (5,6-diepoxydulcitol; NSC132313; CA S Registration number 23261203); fluorodopan ((5-[(2-chloroethyl)-(2-fluoroethyl)amino]-6-methyl-uracil; NSC73754; CAS registration number 834913); hepsulfame (NSC329680; CAS Registration number 96892578); Hikanthone (NSC142982; CAS Registration number 23255938); Melphalan (NSC8806; CAS Registration number 3223072); Methyl CCNU ((1-(2-chloroethyl)-3-(trans-4-methylcyclohexane)-1 - Nitrosourea; NSC95441; 13909096); Mitomycin C (NSC26980; CAS Registry No. 50077); Mitozolamide (NSC353451; CAS Registry No. 85622953); Nitrogen mustard ((bis(2-chloroethyl)methylamine hydrochloride; NSC762) ; CAS registration number 55867); PCNU ((1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea; NSC95466; CAS registration number 13909029)); piperazine alkylating agent ((1-(2-chloroethyl)-4-(3-chloropropyl)-piperazine dihydrochloride; NSC344007)); Piperazinedione (NSC135758; CAS registration number 41109802); Pipobroman ((N,N-bis(3-bromo NSC25154; CAS Registry No. 54911)); porphyromycin (N-methylmitomycin C; NSC56410; CAS Registry No. 801525); spirohydantoin mustard (NSC172112; CAS Registry No. 56605164); teroxirone (triglycidyl Isocyanurate; NSC296934; CAS registration number 2451629); Tetraplatin (NSC363812; CAS registration number 62816982); Thiotepa (N,N',N''-tri-1,2-ethanediylthiophosphoramide; NSC6396; CAS registration No. 52244); Triethylene melamine (NSC9706; CAS registration number 51183); Uracil nitrogen mustard (desmethyldopan; NSC34462; CAS registration number 66751); Yoshi-864 ((bis(3-mesyloxypropyl)amine) Hydrochloride; NSC102627; CAS Registry No. 3458228).

Topoisomerase I inhibitors: camptothecin (NSC94600; CAS Registry No. 7689-03-4); various camptothecin derivatives and analogs (e.g. NSC100880, NSC603071, NSC107124, NSC643833, NSC629971, NSC295500, NSC2499 10, NSC606985, NSC74028, NSC176323, NSC295501 , NSC606172, NSC606173, NSC610458, NSC618939, NSC610457, NSC610459, NSC606499, NSC610456, NSC364830, and NSC606497); morpholinisoxorubicin ( NSC354646; CAS registration number 89196043); SN-38 (NSC673596; CAS registration number 86639) -52-3).

Topoisomerase II inhibitors: doxorubicin (NSC123127; CAS Registry No. 25316409); amonafide (benzoisoquinolinedione; NSC308847; CAS Registry No. 69408817); m-AMSA ((4'-(9-acridinylamino)-3'-methoxy Methanesulfonanilide; NSC249992; CAS registration number 51264143)); anthrapyrazole derivative ((NSC355644); etoposide (VP-16; NSC141540; CAS registration number 33419420); pyrazoloacridine ((pyrazolo[3,4,5-kl] Acridine-2(6H)-propanamine, 9-methoxy-N,N-dimethyl-5-nitro-, monomethanesulfonate; NSC366140; CAS registration number 99009219); bisanthrene hydrochloride (NSC337766; CAS registration number 71439684) Daunorubicin (NSC821151; CAS Registry No. 23541506); Deoxydoxorubicin (NSC267469; CAS Registry No. 63950061); Mitoxantrone (NSC301739; CAS Registry No. 70476823); Menogaril (NSC269148; CAS Registry No. 716289) 61); N,N-dibenzyl Daunomycin (NSC268242; CAS registration number 70878512); oxanthrazole (NSC349174; CAS registration number 105118125); rubidazone (NSC164011; CAS registration number 36508711); teniposide (VM-26; NSC122) 819; CAS registration number 29767202 ).

DNA intercalating agents: Anthramycin (CAS Registry No. 4803274); Ticamycin A (CAS Registry No. 89675376); Tomaymycin (CAS Registry No. 35050556); DC-81 (CAS Registry No. 81307246); Sibiromycin (CAS Registry No. 12684332); Pyrrolobenzodiazepine Derivative (CAS registration number 945490095); SGD-1882 ((S)-2-(4-aminophenyl)-7-methoxy-8-(3-4(S)-7-methoxy-2-(4-methoxyphenyl) )-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-1H-benzo[e]pyrrolo[ 1,2-a][1,4]diazepin-5(11aH)-one);SG2000(SJG-136;(11aS,11a'S)-8,8'-(propane-1,3-diylbis(oxy) )) bis(7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one); NSC694501; CAS Registration number 232931576).

RNA/DNA antimetabolites: L-alanosine (NSC153353; CAS registration number 59163416); 5-azacytidine (NSC102816; CAS registration number 320672); 5-fluorouracil (NSC19893; CAS registration number 51218); Acivicin (NSC163501; C AS registration number 42228922); Aminopterin derivative N-[2-chloro-5-[[(2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino]benzoyl-]L-aspartic acid (NSC132483); Aminopterin derivative N-[4-[[(2,4-diamino-5-ethyl-6-quinazolinyl)methyl]amino]benzoyl]L-aspartic acid (NSC184692); Aminopterin derivative N-[2-chloro-4-[[ (2,4-diamino-6-pteridinyl)methyl]amino]benzoyl]L-aspartic acid monohydrate (NSC134033); antifolate ((N ^α -(4-amino-4-deoxyptero) Baker's soluble antifolate (antifol) (NSC139105; ^CAS registration number 41191042); dichloroallyl lawsone ((2-(3 ,3-dichloroallyl)-3-hydroxy-1,4-naphthoquinone; NSC126771; CAS registration number 36417160); brequinal (NSC368390; CAS registration number 96201886); 2-furyl)-uracil; NSC148958; CAS Registry No. 37076689); 5,6-dihydro-5-azacytidine (NSC264880; CAS Registry No. 62402317); Methotrexate (NSC740; CAS Registry No. 59052); Methotrexate derivatives (N-[[ 4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]-1-naphthalenyl]carbonyl]L-glutamic acid; NSC174121); PALA ((N-(phosphonoacetyl)-L-aspartate; NSC224131; CAS Registry No. 603425565); Pyrazofrine (NSC143095; CAS Registry No. 30868305); Trimetrexate (NSC352122; CAS Registry No. 82952645).

DNA antimetabolites: 3-HP (NSC95678; CAS registration number 3814797); 2'-deoxy-5-fluorouridine (NSC27640; CAS registration number 50919); 5-HP (NSC107392; CAS registration number 19494894); α-TGDR (α-2'-deoxy-6-thioguanosine; NSC71851 CAS registration number 2133815); aphidicoline glycinate (NSC303812; CAS registration number 92802822); ara C (cytosine arabinoside; NSC63878; CAS registration number 69749); 5-aza-2'-deoxycytidine (NSC127716; CAS Registration Number 2353335); β-TGDR (β-2'-deoxy-6-thioguanosine; NSC71261; CAS Registration Number 789617); Cyclocytidine (NSC145668; CAS Registration Number 10212256); Guanazole (NSC1895; CAS Registry No. 1455772); Hydroxyurea (NSC32065; CAS Registry No. 127071); Inosine glycodialdehyde (NSC118994; CAS Registry No. 23590990); Macbecin II (NSC330500; CAS Registry No. 733) 41738); Pyrazolo Imidazole (NSC51143; CAS Registry No. 6714290); Thioguanine (NSC752; CAS Registry No. 154427); Thiopurine (NSC755; CAS Registry No. 50442).

Cell cycle modulators: silibinin (CAS Registry No. 22888-70-6); epigallocatechin gallate (EGCG; CAS Registry No. 989515); procyanidin derivatives (e.g. procyanidin A1 [CAS Registry No. 103883030], procyanidin B1 [CAS Registry No. 20315257], procyanidin B4 [CAS Registry No. 29106512], arecatannin B1 [CAS Registry No. 79763283]); isoflavones (for example, genistein [4% 5,7-trihydroxyisoflavone; CAS Registry No. 446720], daidzein [4 ',7-dihydroxyisoflavone, CAS Registry No. 486668]; Indole-3-carbinol (CAS Registry No. 700061); Quercetin (NSC9219; CAS Registry No. 117395); Estramustine (NSC89201; CAS Registry No. 2998574); Nocodazole ( CAS registration number 31430189); podophyllotoxin (CAS registration number 518285); vinorelbine tartrate (NSC608210; CAS registration number 125317397); cryptophycin (NSC667642; CAS registration number 124689652).

Kinase inhibitors: Afatinib (CAS Registry No. 850140726); Axitinib (CAS Registry No. 319460850); ARRY-438162 (binimetinib) (CAS Registry No. 606143899); Bosutinib (CAS Registry No. 380843754); Cabozantinib (CAS Registry No. 1140) 909483); ceritinib (CAS registration number 1032900256); crizotinib (CAS registration number 877399525); dabrafenib (CAS registration number 1195765457); dasatinib (NSC732517; CAS registration number 302962498); erlotinib (NSC718781; CAS registration number 183319) 699); everolimus (NSC733504; CAS registration number Fostamatinib (NSC745942; CAS Registry No. 901119355); Gefitinib (NSC715055; CAS Registry No. 184475352); Ibrutinib (CAS Registry No. 936563961); Imatinib (NSC716051; CAS Registry No. 22) 0127571); lapatinib (CAS registration number 388082788); lenvatinib (CAS registration number 857890392); Mubritinib (CAS366017096); Nilotinib (CAS registration number 923288953); Nintedanib (CAS registration number 656247175); Palbociclib (CAS registration number 571190302); Pazopanib (NSC737754; CAS registration No. 635702646); Pegaptanib (CAS registered) Ponatinib (CAS Registry No. 1114544318); Rapamycin (NSC226080; CAS Registry No. 53123889); Regorafenib (CAS Registry No. 755037037); AP23573 (Ridaforolimus) (CAS Registry No. 572924540); INC B018424 (Ruxolitinib) (CAS registration) ARRY-142886 (selumetinib) (NSC741078; CAS registration number 606143-52-6); Sirolimus (NSC226080; CAS registration number 53123889); Sorafenib (NSC724772; CAS registration number 475207591); Sunitinib (NSC736511; CAS registration number 341031547); tofacitinib (CAS registration number 477600752); temsirolimus (NSC683864; CAS registration number 163635043); trametinib (CAS registration number 871700173); vandetanib (CAS registration number 443913733); vemurafenib (CAS registration number No. 918504651); SU6656 (CAS registration number CEP-701 (lesaurtinib) (CAS registration number 111358884); XL019 (CAS registration number 945755566); PD-325901 (CAS registration number 391210109); PD-98059 (CAS registration number 167869218) ); ATP competition TORC1 /TORC2 inhibitors, such as PI-103 (CAS registration number 371935749), PP242 (CAS registration number 1092351671), PP30 (CAS registration number 1092788094), Torin 1 (CAS registration number 1222998368), LY294002 (CAS registration number 1544) 47366), XL -147 (CAS registration number 934526893), CAL-120 (CAS registration number 870281348), ETP-45658 (CAS registration number 1198357797), PX866 (CAS registration number 502632668), GDC-0941 (CAS registration number 957054307), BG T226 (CAS Registration number 1245537681), BEZ235 (CAS registration number 915019657), XL-765 (CAS registration number 934493762), etc.

Protein synthesis inhibitors: acriflavin (CAS registration number 65589700); amikacin (NSC177001; CAS registration number 39831555); arbekacin (CAS registration number 51025855); astromycin (CAS registration number 55779061); azithromycin (NSC643732; CAS registration number 8390501) 5) ; Bekanamycin (CAS Registry No. 4696768); Chlortetracycline (NSC13252; CAS Registry No. 64722); Clarithromycin (NSC643733; CAS Registry No. 81103119); Clindamycin (CAS Registry No. 18323449); Chromocycline (CAS Registry No. 1181540) ); cycloheximide (CAS registration number 66819); dactinomycin (NSC3053; CAS registration number 50760); dalfopristin (CAS registration number 112362502); demeclocycline (CAS registration number 127333); dibekacin (CAS registration number 34493986) ; Dihydrostreptomycin (CAS Registry No. 128461); Dirithromycin (CAS Registry No. 62013041); Doxycycline (CAS Registry No. 17086281); Emetine (NSC33669; CAS Registry No. 483181); Erythromycin (NSC55929; CAS Registry No. 114078); Flurithro Mycin (CAS Registry No. 83664208); Framycetin (Neomycin B; CAS Registry No. 119040); Gentamycin (NSC82261; CAS Registry No. 1403663); Glycylcyclines, such as tigecycline (CAS Registry No. 220620097); Hygromycin B (CAS Registry No. Isepamycin (CAS Registry No. 67814760); Josamycin (NSC122223; CAS Registry No. 16846245); Kanamycin (CAS Registry No. 8063078); Ketolides such as telithromycin (CAS Registry No. 191114484), cesthromycin (CAS Registry No. 205110481) , and solithromycin (CAS Registry No. 760981837); lincomycin (CAS Registry No. 154212); limecycline (CAS Registry No. 992212); meclocycline (NSC78502; CAS Registry No. 2013583); metacycline (rondomycin); NSC356463; CAS Registry No. 914001); Midecamycin (CAS Registry No. 35457808); Minocycline (NSC141993; CAS Registry No. 10118908); Myocamycin (CAS Registry No. 55881077); Neomycin (CAS Registry No. 119040); Netilmicin (CAS Registry No. 56) 391561) ; oleandomycin (CAS Registry No. 3922905); oxazolidinones such as eperezolid (CAS Registry No. 165800044), linezolid (CAS Registry No. 165800033), posizolid (CAS Registry No. 252260029), radezolid (CAS Registry No. 869884786) ), lambezolid (ranbezolid) (CAS Registry No. 392659380), Stezolid (CAS Registry No. 168828588), Tedizolid (CAS Registry No. 856867555); Oxytetracycline (NSC9169; CAS Registry No. 2058460); Paromomycin (CAS Registry No. 7542372); Penimepicycline ( CAS Registry No. 4599604); peptidyltransferase inhibitors, such as chloramphenicol (NSC 3069; CAS Registry No. 56757) and azidamphenicol (CAS Registry No. 13838089), florfenicol (CAS Registry No. 73231342), and thiamphenicol (CAS Registry No. 4599604); Cole (CAS Registry No. 15318453), and derivatives of ploiromutilins such as retapamulin (CAS Registry No. 224452668), tiamulin (CAS Registry No. 55297955), valnemulin (CAS Registry No. 101312929); pirrimycin (CAS Registry No. 79548735); puromycin ( NSC3055; CAS registration number 53792); Quinupristin (CAS registration number 120138503); Ribostamycin (CAS registration number 53797356); Rokitamycin (CAS registration number 74014510); Rolitetracycline (CAS registration number 751973); Roxithromycin (CAS registration number) 80214831); Sisomicin (CAS Registry No. 32385118); Spectinomycin (CAS Registry No. 1695778); Spiramycin (CAS Registry No. 8025818); Streptogramins such as pristinamycin (CAS Registry No. 270076603), Quinupristin/Dalfopristin (CAS Registry No. 270076603); Streptomycin (CAS Registry No. 57921); Tetracycline (NSC108579; CAS Registry No. 60548); Tobramycin (CAS Registry No. 32986564); Troleandomycin (CAS Registry No. 2751099); Tylosin (CAS Registry No. 1401690); Verdamicin (CAS Registry No. 49863481).

Histone deacetylase inhibitors: Abexinostat (CAS Registry No. 783355602); Belinostat (NSC726630; CAS Registry No. 414864009); Chidamide (CAS Registry No. 743420022); Entinostat (CAS Registry No. 209783802); Gibinostat ( givinostat) (CAS Registration Number 732302997); Mosetinostat (CAS Registration Number 726169739); Panobinostat (CAS Registration Number 404950807); Xinostat (CAS Registration Number 875320299); Resminostat (CAS Registration Number 864814880); 128517077 ); Sulforaphane (CAS Registry No. 4478937); Thiouridobutyronitrile (Kevetrin™; CAS Registry No. 6659890); Valproic acid (NSC93819; CAS Registry No. 99661); Vorinostat (NSC701852; CAS Registry No. 149647789); ACY -1215 (rocilinostat; CAS registration number 1316214524); CUDC-101 (CAS registration number 1012054599); CHR-2845 (tefinostat; CAS registration number 914382608); CHR-3996 (CAS registration number 1235859138); 4SC-202 (CAS registration number 910462430); CG200745 (CAS registration number 936221339); SB939 (prasinostat; CAS registration number 929016966).

Mitochondrial inhibitor: Punk Latty Statin (NSC349156; CAS Registration Number 96281311); Rodamin -123 (CAS registration number 63669709); Edelfosine (NSC324668; CAS Registration number 70641519); D -alpha -cocoquettopoferolol (NSC173849; CAS Compound 11β (CAS Registry No. 865070377); Aspirin (NSC406186; CAS Registry No. 50782); Ellipticine (CAS Registry No. 519233); Berberine (CAS Registry No. 633658); Cerulenin (CAS Registry No. 17397896); GX015- 070 (Obatoclax®; 1H-indole, 2-(2-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-; NSC729280; CAS registration number 803712676); Celastrol (tripterin; CAS registration number 34157830); Metformin (NSC91485; CAS registration number 1115704); Brilliant Green (NSC5011; CAS registration number 633034); ME-344 (CAS registration number 13745) 24556).

Antimitotic agents: allocolchicine (NSC406042); auristatins, such as MMAE (monomethyl auristatin E; CAS Registry No. 474645-27-7) and MMAF (monomethyl auristatin F; CAS Registry No. 745017-94-) 1; halichondrin B (NSC609395); colchicine (NSC757; CAS registration number 64868); colchicine derivative (N-benzoyl-deacetylbenzamide; NSC33410; CAS registration number 63989753); dolastatin 10 (NSC376128; CAS registration number 110417) -88-4); maytansine (NSC153858; CAS registration number 35846-53-8); rhozoxin (NSC332598; CAS registration number 90996546); taxol (NSC125973; CAS registration number 33069624); taxol derivatives ((2'- N-[3-(dimethylamino)propyl]glutaramate taxol; NSC608832); Thiocolchicine (3-demethylthiocolchicine; NSC361792); Tritylcysteine (NSC49842; CAS registration number 2799077); Vinblastine sulfate (NSC49842; CAS registration No. 143679); vincristine sulfate (NSC67574; CAS Registry No. 2068782).

Any of these agents that contain or can be modified to contain an attachment site for antibodies can be included in the ADCs disclosed herein.

In specific embodiments, the cytotoxic and/or cytostatic agent is an antimitotic agent.

In another specific embodiment, the cytotoxic and/or cytostatic agent is an auristatin, such as monomethyl auristatin E ("MMAE") or monomethyl auristatin F ("MMAF").

5.3.2. Linker In the anti-glycoMUC1 ADC of the present disclosure, the cytotoxic and/or cytostatic agent is linked to the antibody by a linker. The linker that connects the cytotoxic and/or cytostatic agent to the antibody of the ADC may be a short, long, hydrophobic, hydrophilic, flexible, or rigid linker, or the linker may have different properties. It may be composed of segments each independently having one or more of the above-described characteristics, as may include segments having one or more of the above-described characteristics. Linkers can be multivalent such that they covalently link more than one drug to a single site on an antibody, or they link a single drug to a single site on an antibody. It may be monovalent so that it is covalently linked to the site.

As one of skill in the art would understand, the linker forms a covalent linkage to the cytotoxic and/or cytostatic agent at one position and to the antibody at the other position. The cytotoxic and/or cytostatic agent is linked to the antibody by forming a covalent linkage. Covalent linkages are formed by reaction of functional groups on the linker with functional groups on the drug and antibody. As used herein, the expression "linker" refers to (i) a functional group capable of covalently linking the linker to a cytotoxic and/or cytostatic agent; (ii) a functional group capable of covalently linking the linker to the antibody; (ii) the linker is capable of covalently linking the linker to the antibody; a partially conjugated form of a linker that includes a functional group covalently linked to a cytostatic agent, or vice versa; and (iii) a cytotoxic agent and/or a cytostatic agent. It is intended to include fully conjugated forms of linkers that are covalently linked to both the substance and the antibody. In addition to the linkers and anti-glyco-MUC1 ADCs of the present disclosure, in some specific embodiments of the linker-synthons used to conjugate drugs to antibodies, functional groups on the linker and Moieties containing covalent bonds formed therebetween are specifically exemplified as R _x and XY, respectively.

The linker is preferably, but not necessarily, chemically stable to conditions outside the cell and cannot be cleaved, disrupted, and/or otherwise inside the cell. may be designed to be specifically degraded by Alternatively, linkers that are not designed to be specifically cleaved or degraded inside the cell may be used. The choice between stable and unstable linkers may depend on the toxicity of the cytotoxic and/or cytostatic agent. For drugs that are toxic to normal cells, stable linkers are preferred. Agents that are selective for or target normal cells to reduce toxicity may be utilized, and the chemical stability of the linker relative to the extracellular environment is less important. A variety of linkers useful for linking drugs to antibodies in ADCs are known in the art. Any of these linkers, and other linkers, can be used to link cytotoxic and/or cytostatic agents to the antibodies of the anti-glycoMUC1 ADCs of the present disclosure.

Exemplary multivalent linkers that can be used to link multiple cytotoxic and/or cytostatic agents to a single antibody molecule are described, e.g., the contents of which are incorporated herein by reference in their entirety. International Publication No. 2009/073445 pamphlet; International Publication No. 2010/068795 pamphlet; International Publication No. 2010/138719 pamphlet; International Publication No. 2011/120053 pamphlet; International Publication No. 2011/171020 pamphlet; International Publication No. It is described in International Publication No. 2013/096901 pamphlet; International Publication No. 2014/008375 pamphlet; International Publication No. 2014/093379 pamphlet; International Publication No. 2014/093394 pamphlet; International Publication No. 2014/093640 pamphlet. For example, the Fleximer linker technology developed by Mersana et al. has the ability to enable high DAR ADCs with excellent physicochemical properties. As shown below, Mersana technology is based on incorporating drug molecules into a soluble polyacetal backbone through an array of ester bonds. This approach results in highly loaded ADCs (up to 20 DAR) while maintaining excellent physicochemical properties.

Further examples of dendritic linkers are found in U.S. Patent Application Publication No. 2006/116422; U.S. Patent Application Publication No. 2005/271615; de Groot et al. 2003) Angew. Chem. Int. Ed. 42:4490-4494; Amir et al. (2003) Angew. Chem. Int. Ed. 42:4494-4499; Shamis et al. (2004) J. Am. Chem. Soc. 126:1726-1731; Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11:1761-1768; King et al. (2002) Tetrahedron Letters 43:1987-1990.

Exemplary monovalent linkers that can be used include, for example, Nolting, 2013, Antibody-Drug Conjugates, Methods in Molecular Biology 1045:71-100; Kitson et al., 2013, CROs, each of which is incorporated herein by reference. /CMOs--Chemica Oggi--Chemistry Today 31(4):30-38; Ducry et al., 2010, Bioconjugate Chem. 21:5-13; Zhao et al., 2011, J. Med. Chem. 54: 3606-3623; US Pat. No. 7,223,837; US Pat. No. 8,568,728; US Pat. No. 8,535,678; and WO 2004010957 pamphlet. ing.

By way of example, but not limitation, some cleavable and non-cleavable linkers that may be included in the anti-glyco-MUC1 ADCs of the present disclosure are described below.

5.3.3. Cleavable Linkers In certain embodiments, the selected linker is cleavable in vivo. Cleavable linkers may include chemically or enzymatically labile or degradable linkages. Cleavable linkers are generally used to release the drug due to processes internal to the cell, such as reduction within the cytoplasm, exposure to acidic conditions in lysosomes, or by specific proteases or other enzymes within the cell. Rely on amputation. A cleavable linker generally incorporates one or more chemical bonds that are cleavable either chemically or enzymatically while the remainder of the linker is non-cleavable. In certain embodiments, the linker includes chemically labile groups, such as hydrazone and/or disulfide groups. Linkers containing chemically labile groups take advantage of the different properties between plasma and some intracytoplasmic compartments. The intracellular conditions that facilitate drug release in the case of hydrazone-containing linkers are the acidic environments of endosomes and lysosomes, whereas disulfide-containing linkers are preferred in the cytosol containing high thiol concentrations, e.g. glutathione. will be returned. In certain embodiments, the plasma stability of linkers that include chemically labile groups can be increased by introducing steric hindrance using substituents near the chemically labile groups.

Acid-labile groups, such as hydrazone, remain intact during systemic circulation in the neutral pH environment of the blood (pH 7.3-7.5), whereas ADCs are found in the slightly acidic endosomes of cells (pH 5. 0-6.5) and lysosomal (pH 4.5-5.0), it undergoes hydrolysis and releases the drug. This pH-dependent release mechanism is associated with non-specific release of drug. To increase the stability of the hydrazone group in the linker, the linker can be modified by chemical modifications, e.g. substitutions, that can be tailored to achieve more efficient release in lysosomes while minimizing losses in the circulation. You can.

The hydrazone-containing linker may contain additional cleavage sites, such as additional acid-labile cleavage sites and/or enzyme-labile cleavage sites. An exemplary hydrazone-containing linker-containing ADC includes the following structure.

where D and Ab represent a cytotoxic and/or cytostatic substance (drug) and Ab, respectively, and n represents the number of drug-linkers linked to the antibody. In certain linkers, such as linker (Ig), the linker contains two cleavable groups, a disulfide moiety and a hydrazone moiety. For such linkers, effective release of unmodified free drug requires acidic pH or disulfide reduction and acidic pH. Linkers such as (Ih) and (Ii) have been shown to be effective at single hydrazone cleavage sites.

Additional linkers that remain intact during systemic circulation but undergo hydrolysis to release the drug once the ADC is internalized inside acidic cellular compartments include carbonates. Such linkers may be useful if a cytotoxic and/or cytostatic agent can be covalently attached via oxygen.

Other acid-labile groups that may be included in the linker include linkers containing cis-aconityl. Cis-aconithyl chemistry uses a carboxylic acid juxtaposed to the amide bond to promote amide hydrolysis under acidic conditions.

Cleavable linkers can also include disulfide groups. Disulfides are thermodynamically stable at physiological pH and are designed to release drugs when internalized inside the cell, where the cytosol is more significantly compared to the extracellular environment. provide a highly reducing environment for The dissociation of disulfide bonds is generally achieved by reducing cytoplasmic thiol cofactors, e.g. ) requires the presence of glutathione (GSH). Also, the intracellular enzyme protein disulfide isomerase, or similar enzymes capable of cleaving disulfide bonds, may also contribute to the preferential cleavage of disulfide bonds inside the cell. In approximately 5 tumor cells, GSH has a concentration range of 0.5-10 mM compared to significantly lower concentrations of GSH or cysteine (which is the most abundant low molecular weight thiol) in the circulation. It has been reported that glutathione is present in cells, where irregular blood flow causes hypoxia and enhances the activity of reducing enzymes, thus resulting in even higher glutathione concentrations. In certain embodiments, the in vivo stability of disulfide-containing linkers can be enhanced by chemical modification of the linker, eg, by the use of steric hindrance adjacent to the disulfide bond.

An exemplary disulfide-containing linker-containing ADC includes the following structure.

where D and Ab represent drug and antibody, respectively, n represents the number of drug-linkers attached to the antibody, and R is independently selected at each occurrence from, for example, hydrogen or alkyl. In certain embodiments, increasing the steric hindrance adjacent to the disulfide bond increases the stability of the linker. Structures such as (Ij) and (Il) exhibit increased stability in vivo when one or more R groups are selected from lower alkyl, for example methyl.

Another type of cleavable linker that can be used is a linker that is specifically cleaved by an enzyme. Such linkers are typically peptide-based or include a peptide region that acts as a substrate for the enzyme. Peptide-based linkers tend to be more stable in the plasma and extracellular environment than chemically labile linkers. Lysosomal proteolytic enzymes have very low activity in the blood due to endogenous inhibitors and the unfavorably high pH value of blood compared to lysosomes, so peptide bonds are generally superior in serum. It has excellent stability. Drug release from antibodies specifically occurs due to the action of lysosomal proteases such as cathepsins and plasmin. These proteases may be present at high levels in certain tumor cells.

In an exemplary embodiment, the cleavable peptide is a tetrapeptide such as Gly-Phe-Leu-Gly (SEQ ID NO: 128), Ala-Leu-Ala-Leu (SEQ ID NO: 129), or Val-Cit, Val- Ala, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, Phe-Lys, Ile-Val, Asp-Val, His-Val, NorVal-(D)Asp, Ala-(D ) Asp5, Met-Lys, Asn-Lys, Ile-Pro, Me3Lys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met- selected from dipeptides such as (D)Lys, Asn-(D)Lys, AM Met-(D)Lys, Asn-(D)Lys, AW Met-(D)Lys, and Asn-(D)Lys. In certain embodiments, dipeptides are preferred over longer polypeptides because longer peptides are hydrophobic.

A variety of dipeptide-based cleavable linkers have been described that are useful for linking drugs to antibodies, such as doxorubicin, mitomycin, camptothecin, pyrrolobenzodiazepines, tallysomycin, and auristatin/auristatin family members (each with Dubowchik et al., 1998, J. Org. Chem. 67:1866-1872; Dubowchik et al., 1998, Bioorg. Med. Chem. Lett. 8(21):3341-, incorporated herein by reference. 3346; Walker et al., 2002, Bioorg. Med. Chem. Lett. 12:217-219; Walker et al., 2004, Bioorg. Med. Chem. Lett. 14:4323-4327; Sutherland et al., 2013 , Blood 122: 1455-1463; and Francisco et al., 2003, Blood 102:1458-1465). All of these dipeptide linkers, or modified versions of these dipeptide linkers, can be used in the anti-glyco-MUC1 ADCs of the present disclosure. Other dipeptide linkers that can be used include Seattle Genetics' brentuximab vedotin SGN-35 (Adcetris™), Seattle Genetics' SGN-75 (anti-CD-70, Val-Cit-monomethyl auristatin F (MMAF), SEATTLE GENETICS SGN -CD33A (anti -CD -33, VAL -ALA (SGD -1882)), CELLDEX THERAPEUTICS Glenbatsum Mab (CDX -011) (anti -NMB, VAL -CI T -Monomethyl Ari Statin E (MMAE) ), and those found in ADCs such as Cytogen PSMA-ADC (PSMA-ADC-1301) (anti-PSMA, Val-Cit-MMAE).

Enzymatically cleavable linkers can include self-immolative spacers to spatially separate the drug from the enzymatic cleavage site. Direct attachment of a drug to a peptide linker can cause release of the drug's amino acid adducts by proteolysis, thereby impairing its activity. The use of a self-immolative spacer allows for the elimination of a fully active, chemically unmodified drug after hydrolysis of the amide bond.

One self-immolative spacer is the bifunctional para-aminobenzyl alcohol group, which is linked to the peptide through the amino group to form an amide bond, whereas the amine-containing drug is The benzyl hydroxyl group may be attached via a carbamic acid functionality. The resulting prodrug is activated by protease-mediated cleavage, causing a 1,6 elimination reaction and releasing the remainder of the unmodified drug, carbon dioxide, and linker group. The scheme below depicts the fragmentation of p-amidobenzyl ether and drug release.

where XD represents unmodified drug.

Heterocyclic variants of this self-destructive group have also been described. See, eg, US Pat. No. 7,989,434, which is incorporated herein by reference.

In some embodiments, the enzymatically cleavable linker is a β-glucuronic acid-based linker. Facile release of drug can be achieved through cleavage of β-glucuronide glycosidic bonds by the lysosomal enzyme β-glucuronidase. This enzyme is abundant in lysosomes and overexpressed in some tumor types, but enzyme activity outside the cell is low. β-glucuronic acid-based linkers can be used to avoid the tendency of ADCs to undergo aggregation due to the hydrophilic nature of β-glucuronide. In some embodiments, β-glucuronic acid-based linkers are preferred as linkers for ADCs linked to hydrophobic drugs. The scheme below depicts drug release from an ADC containing a β-glucuronic acid-based linker.

A variety of cleavable β-glucuronic acid-based linkers have been described that are useful for linking drugs to antibodies, such as auristatin, camptothecin and doxorubicin analogs, CBI minor groove binders, and psymberin ( Nolting, Chapter 5 “Linker Technology in Antibody-Drug Conjugates,” In: Antibody-Drug Conjugates: Methods in Molecular Biology, vol. 1045, pp. 71-100, Laurent Ducry ( Ed.), Springer Science & Business Medica, LLC, 2013; Jeffrey et al., 2006, Bioconjug. Chem. 17:831-840; Jeffrey et al., 2007, Bioorg. Med. Chem. Lett. 17:2278- 2280; and Jiang et al., 2005, J. Am. Chem. Soc. 127:11254-11255). All of these β-glucuronic acid-based linkers can be used in the anti-glyco-MUC1 ADCs of the present disclosure.

Additionally, cytotoxic and/or cytostatic substances containing phenolic groups can be covalently attached to the linker via the phenolic oxygen. One such linker is described in WO 2007/089149, which uses diamino-ethane "SpaceLink" with a conventional "PABO"-based self-destructive group to deliver phenol. rely on Cleavage of the linker is schematically depicted below, where D represents a cytotoxic and/or cytostatic substance with a phenolic hydroxyl group.

The cleavable linker may include a non-cleavable portion or segment, and/or the cleavable segment or portion may be inserted into an otherwise non-cleavable linker to make it cleavable. may be included in By way of example only, polyethylene glycol (PEG) and related polymers may include cleavable groups in the polymer backbone. For example, the polyethylene glycol or polymer linker may contain one or more cleavable groups, such as disulfides, hydrazones or dipeptides.

Other degradable linkages that may be included in the linker include ester linkages formed by reaction of the carboxylic acid of PEG or the carboxylic acid of activated PEG with the alcohol group on the bioactive agent; In this case, such ester groups typically hydrolyze under physiological conditions to release the bioactive agent. Hydrolytically degradable linkages include, but are not limited to, carbonate linkages; imine linkages resulting from the reaction of amines and aldehydes; phosphate ester linkages formed by reacting alcohols with phosphate groups; Acetal linkages, which are the reaction products of alcohols; orthoester linkages, which are the reaction products of formates and alcohols; and linkages formed by, but not limited to, phosphoramidite groups at the ends of polymers, and 5' hydroxyl groups of oligonucleotides. oligonucleotide linkage.

In certain embodiments, the linker is an enzymatically cleavable peptide moiety, e.g., structural formula (IVa) or (IVb):

or a salt thereof, where peptide represents a peptide (exemplified by C→N, with carboxy and amino "termini" not shown) that is cleavable by lysosomal enzymes; T is one or represents a polymer comprising multiple ethylene glycol units or alkylene chains, or combinations thereof; R ^a is selected from hydrogen, alkyl, sulfonate and methylsulfonate; p is an integer ranging from 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1;

represents the point of attachment of the linker to the cytotoxic and/or cytostatic agent; ^* represents the point of attachment to the remainder of the linker.

In certain embodiments, the peptide is selected from a tripeptide or a dipeptide. In certain embodiments, the dipeptide is Val-Cit;Cit-Val;Ala-Ala;Ala-Cit;Cit-Ala;Asn-Cit;Cit-Asn;Cit-Cit;Val-Glu;Glu-Val;Ser -Cit;Cit-Ser;Lys-Cit;Cit-Lys;Asp-Cit;Cit-Asp;Ala-Val;Val-Ala;Phe-Lys;Val-Lys;Ala-Lys;Phe-Cit;Leu-Cit ; Ile-Cit; Phe-Arg; and Trp-Cit. In certain embodiments, the dipeptide is selected from Cit-Val; and Ala-Val.

Certain exemplary embodiments of linkers according to structural formula (IVa) that may be included in the anti-glyco MUC1 ADCs of the present disclosure include the linkers exemplified below (as exemplified, the linkers are (containing a group suitable for covalently linking an antibody to an antibody):

Certain exemplary embodiments of linkers according to structural formula (IVb) that may be included in the anti-glyco MUC1 ADCs of the present disclosure include the linkers exemplified below (as exemplified, the linkers are (containing a group suitable for covalently linking an antibody to an antibody):

In certain embodiments, the linker is an enzymatically cleavable peptide moiety, e.g., structural formula (IVc) or (IVd):

or a salt thereof, where peptide represents a peptide (exemplified by C→N, with carboxy and amino "termini" not shown) that is cleavable by lysosomal enzymes; T is one or represents a polymer comprising multiple ethylene glycol units or alkylene chains, or combinations thereof; R ^a is selected from hydrogen, alkyl, sulfonate and methylsulfonate; p is an integer ranging from 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1;

represents the point of attachment of the linker to the cytotoxic and/or cytostatic agent; ^* represents the point of attachment to the remainder of the linker.

Certain exemplary embodiments of linkers according to structural formula (IVc) that may be included in the anti-glyco MUC1 ADCs of the present disclosure include the linkers exemplified below (as exemplified, the linkers are (containing a group suitable for covalently linking an antibody to an antibody):

Certain exemplary embodiments of linkers according to structural formula (IVd) that may be included in the anti-glyco MUC1 ADCs of the present disclosure include the linkers exemplified below (as exemplified, the linkers are (containing a group suitable for covalently linking an antibody to an antibody):

In certain embodiments, a linker comprising structural formula (IVa), (IVb), (IVc), or (IVd) further comprises a carbonate moiety that is cleavable by exposure to acidic media. In certain embodiments, the linker is attached to the cytotoxic and/or cytostatic agent via oxygen.

5.3.4. Non-cleavable Linkers Although cleavable linkers may provide certain advantages, the linkers comprising the anti-glyco-MUC1 ADCs of the present disclosure do not necessarily have to be cleavable. In the case of non-cleavable linkers, drug release does not depend on different properties between plasma and some intracytoplasmic compartment. Drug release is hypothesized to occur after internalization of the ADC via antigen-mediated endocytosis and delivery to the lysosomal compartment, where the antibody is degraded to amino acid levels via intracellular proteolysis. This process releases a drug derivative formed by the drug, the linker, and the amino acid residue to which the linker is covalently attached. Amino acid drug metabolites from conjugates with non-cleavable linkers are more hydrophilic and generally have lower membrane permeability compared to conjugates with cleavable linkers, thereby , fewer bystander effects and less non-specific toxicity result. Generally, ADCs with non-cleavable linkers have greater stability in circulation than ADCs with cleavable linkers. The non-cleavable linker may be an alkylene chain or may be based on a natural polymer, such as a polyalkylene glycol polymer, an amide polymer, or an alkylene chain, a polyalkylene glycol (glocol). and/or amide polymer segments.

A variety of non-cleavable linkers have been described that are used to link drugs to antibodies. Jeffrey et al., 2006, Bioconjug. Chem. 17; 831-840; Jeffrey et al., 2007, Bioorg. Med. Chem. Lett. 17:2278-2280; and See Jiang et al., 2005, J. Am. Chem. Soc. 127:11254-11255. Any of these linkers may be included in the anti-glycoMUC1 ADCs of the present disclosure.

In certain embodiments, the linker is non-cleavable in vivo, e.g., a linker according to structural formula (VIa), (VIb), (VIc) or (VId) (as exemplified), and the linker is Suitable groups for covalently linking a linker to an antibody:

or a salt thereof, wherein R ^a is selected from hydrogen, alkyl, sulfonate and methylsulfonate; R ^x is a moiety containing a functional group capable of covalently linking the linker to the antibody;

represents the point of attachment of the linker to the cytotoxic and/or cytostatic agent.

Certain exemplary embodiments of linkers according to structural formulas (VIa)-(VId) that may be included in the anti-glyco-MUC1 ADCs of the present disclosure include the linkers exemplified below (as exemplified). the linker comprises a group suitable for covalently linking the linker to the antibody;

represents the point of attachment to cytotoxic and/or cytostatic substances).

5.3.5. Groups Used to Attach Linkers to Antibodies A variety of groups can be used to attach linkers--drug synthons to antibodies to obtain ADCs. The attachment group may be electrophilic in nature, examples being maleimide groups, activated disulfides, active esters such as NHS esters and HOBt esters, formate halides, acid halides, alkyl halides and Mention may be made of benzyl, such as haloacetamide. As discussed below, there is also emerging technology regarding "self-stabilizing" maleimides and "bridge-forming disulfides" that can be used in accordance with the present disclosure. The specific group used will depend in part on the site of attachment to the antibody.

An example of a "self-stabilizing" maleimide group that spontaneously hydrolyzes under antibody conjugation conditions to yield an ADC species with improved stability is depicted in the schematic below. See also US Patent Application Publication No. 20130309256; also Lyon et al., Nature Biotech published online, doi:10.1038/nbt.2968.

Polytherics discloses a method for crosslinking pairs of sulfhydryl groups derived from the reduction of natural hinge disulfide bonds. See Badescu et al., 2014, Bioconjugate Chem. 25:1124-1136. This reaction is depicted in the schematic diagram below. The advantage of this approach is the ability to synthesize enriched DAR4 ADCs by complete reduction of IgG (resulting in four pairs of sulfhydryls) followed by reaction with four equivalents of alkylating agent. ADCs containing "crosslinked disulfides" are also said to have increased stability.

Similarly, maleimide derivatives (1, below) have been developed that are capable of cross-linking pairs of sulfhydryl groups, as depicted below. Please refer to International Publication No. 2013/085925 pamphlet.

5.3.6. Linker Selection Considerations As known to those skilled in the art, the selected linker for a particular ADC may include, but is not limited to, the site of attachment to the antibody (e.g., lys, cys or other amino acid residues), the drug may be influenced by various factors such as the structural constraints of the pharmacophore, and the lipophilicity of the drug. The selected linker for a particular ADC will be required to balance these various factors for the particular antibody/conjugate. For a review of factors influenced by linker selection in ADCs, see Nolting, Chapter 5 “Linker Technology in Antibody-Drug Conjugates,” In: Antibody-Drug Conjugates: Methods in Molecular Biology, vol. 1045, pp. 71-100, See Laurent Ducry (Ed.), Springer Science & Business Medica, LLC, 2013.

For example, ADCs have been observed to kill bystander antigen-negative cells that reside near antigen-positive tumor cells. The mechanism of bystander cell killing by ADCs indicated that metabolites formed during intracellular processing of ADCs may play a role. Neutral cytotoxic metabolites generated by ADC metabolism in antigen-positive cells appear to play a role in bystander cell killing, whereas charged metabolites are more likely to enter the media across the membrane. Spread may be blocked and therefore bystander lethality cannot be affected. In certain embodiments, the linker is selected to attenuate bystander lethal effects caused by cellular metabolites of the ADC. In certain embodiments, the linker is selected to increase bystander lethality.

The properties of the linker may also affect ADC aggregation under conditions of use and/or storage. Typically, ADCs reported in the literature contain no more than 3-4 drug molecules per antibody molecule (see, eg, Chari, 2008, Acc Chem Res 41:98-107). Attempts to obtain higher drug-to-antibody ratios (“DAR”) often fail due to ADC aggregation, especially when both drug and linker are hydrophobic (King et al., 2002, J Med Chem 45:4336-4343; Hollander et al., 2008, Bioconjugate Chem 19:358-361; Burke et al., 2009 Bioconjugate Chem 20:1242-1250). In many instances, a DAR higher than 3-4 can be beneficial as a means of increasing efficacy. In instances where the cytotoxic and/or cytostatic agent is hydrophobic in nature, relatively hydrophilic linkers may be used as a means of reducing ADC aggregation, particularly in instances where a DAR greater than 3-4 is desired. It may be desirable to select Thus, in certain embodiments, the linker incorporates chemical moieties that reduce aggregation of the ADC during storage and/or use. The linker may incorporate polar or hydrophilic groups, such as charged groups or groups that become charged at physiological pH, to reduce aggregation of the ADC. For example, the linker may incorporate a charged group, such as a salt or group, that deprotonates, for example, a carboxylic acid, or protonates, for example, an amine, at physiological pH.

Exemplary multivalent linkers that can be used to link numerous cytotoxic and/or cytostatic substances to antibodies, which have been reported to yield DARs as high as 20, are WO 2009/073445 brochure; WO 2010/068795 brochure; WO 2010/138719 brochure; WO 2011/120053 brochure; WO 2011/ 171020 pamphlet; International Publication No. 2013/096901 pamphlet; International Publication No. 2014/008375 pamphlet; International Publication No. 2014/093379 pamphlet; International Publication No. 2014/093394 pamphlet; Described in International Publication No. 2014/093640 pamphlet has been done.

In certain embodiments, the aggregation of the ADC during storage or use is less than about 10% as determined by size exclusion chromatography (SEC). In certain embodiments, the aggregation of the ADC during storage or use is less than 10%, such as less than about 5%, less than about 4%, less than about 3%, about less than 2%, less than about 1%, less than about 0.5%, less than about 0.1%, or even lower.

5.3.7. Methods of Making Anti-Glyco-MUC1 ADCs The anti-glyco-MUC1 ADCs of the present disclosure can be synthesized using well-known chemical methods. The chemical method chosen will depend, among other things, on the nature of the cytotoxic and/or cytostatic agent, the linker and group used to attach the linker to the antibody. Generally, ADCs according to formula (I) can be prepared according to the following scheme:
D-L-R ^x +Ab-R ^y → [DL-XY] _n -Ab (I)
where ^D , L, ^Ab , represents a group.

The nature of the R ^x and R ^y groups is expected to depend on the chemical method used to link the synthon DLR ^x to the antibody. Generally, the chemical method used should not alter the integrity of the antibody, eg, its ability to bind to its target. Preferably, the binding properties of the conjugated antibody are expected to be very similar to those of the unconjugated antibody. A variety of chemical methods and techniques are known in the art for conjugating molecules to biomolecules such as antibodies, particularly those for conjugating to antibodies. For example, Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in: Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. Eds., Alan R. Liss, Inc., 1985; Hellstrom et al., “ Antibodies For Drug Delivery,” in: Controlled Drug Delivery, Robinson et al. Eds., Marcel Dekker, Inc., 2nd Ed. 1987; Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in: Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al., Eds., 1985; “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy,” in: Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al., Eds., Academic Press, 1985; Thorpe et al., 1982, Immunol. Rev. 62:119-58; see PCT International Publication No. 89/12624 pamphlet. Any of these chemical methods can be used to link synthons to antibodies.

The number of functional groups R ^x and chemical methods useful for linking synthons to accessible lysine residues are known and include, but are not limited to, NHS-esters and isothiocyanates.

The number of functional groups R ^x and chemical methods useful for linking synthons to accessible free sulfhydryl groups of cysteine residues are known, and examples include, but are not limited to, haloacetyl and maleimide. It will be done.

However, conjugation chemistry is not limited to the available side groups. Side chains such as amines can be converted to other useful groups, such as hydroxyl, by linking appropriate small molecules to the amine. This strategy can be used to increase the number of available linking sites on antibodies by conjugating multifunctional small molecules to the side chains of accessible amino acid residues of antibodies. Functional groups R ^x suitable for covalently linking the synthon to these "converted" functional groups are then included in the synthon.

Antibodies can also be engineered to include amino acid residues for conjugation. Approaches for engineering antibodies to include non-genetically encoded amino acid residues useful for conjugating drugs in the form of ADCs include chemical methods and chemical methods useful for linking synthons to non-encoded amino acids. along with the functional groups described by Axupet al., 2012, Proc Natl Acad Sci USA. 109(40):16101-16106.

Typically, the synthon is linked to the side chain of an amino acid residue of the antibody, such as the primary amino group of an accessible lysine residue or the sulfhydryl group of an accessible cysteine residue. Free sulfhydryl groups can be obtained by reducing interchain disulfide bonds.

For linkages where R ^y is a sulfhydryl group (e.g., R ^x is maleimide), the antibody is generally first fully or partially reduced to remove the interchain disulfide bridges between cysteine residues. collapse.

Cysteine residues that do not participate in disulfide bridges can be engineered into the antibody by mutation of one or more codons. Reduction of these unpaired cysteines yields sulfhydryl groups suitable for conjugation. Preferred positions for incorporating engineered cysteines include, by way of example and without limitation, positions S112C, S113C, A114C, S115C, A176C, 5180C, and S252C on the human IgG ₁ heavy chain. , V286C, V292C, S357C, A359C, S398C, S428C (Kabat numbering), and V110C, S114C, S121C, S127C, S168C, V205C on the human Ig kappa light chain. (Kabat numbering) (see, e.g., U.S. Patent No. 7,521,541, U.S. Patent No. 7,855,275, and U.S. Patent No. 8,455,622). ).

As would be expected to be understood by those skilled in the art, multiple cytotoxic and/or cytostatic agents may be linked to an antibody molecule; some antibodies contain one linked agent. , some antibodies contain two linked drugs, some antibodies contain three linked drugs, etc. (some antibodies contain no linked drugs), etc. may be heterogeneous in nature. The degree of heterogeneity is expected to depend, among other things, on the chemical method used to link the cytotoxic and/or cytostatic agent. For example, when antibodies are reduced to generate sulfhydryl groups for attachment, a heterogeneous mixture of antibodies with 0, 2, 4, 6, or 8 linked drugs per molecule can be produced. many. Furthermore, by limiting the molar ratio of the attachment compounds, antibodies with 0, 1, 2, 3, 4, 5, 6, 7 or 8 linked drugs per molecule are often produced. . It is therefore expected that, depending on the context, the stated DAR may be an average of a population of antibodies. For example, "DAR4" has not been subjected to purification to isolate specific DAR peaks and has different numbers of cytostatic and/or cytotoxic substances attached per antibody ( can contain a heterogeneous mixture of ADC molecules (e.g., 0, 2, 4, 6, 8 drugs per antibody), but can refer to an ADC preparation in which the average drug-to-antibody ratio is 4. . Similarly, in some embodiments, "DAR2" refers to a heterogeneous ADC preparation in which the average drug-to-antibody ratio is 2.

If an enriched preparation is desired, antibodies with a defined number of linked cytotoxic and/or cytostatic substances can be purified via purification of the heterogeneous mixture, e.g. by column chromatography, e.g. , can be obtained via hydrophobic interaction chromatography.

Purity can be assessed by various methods as known in the art. As a specific example, ADC preparations may be analyzed via HPLC or other chromatography, and purity may be assessed by analyzing the area under the curve of the resulting peaks.

5.4 Chimeric Antigen Receptors The present disclosure provides chimeric antigen receptors (CARs) comprising anti-glycoMUC1 antibodies or antigen binding fragments described herein.

The CARs of the present disclosure typically include an extracellular domain operably linked to a transmembrane domain, which in turn is operably linked to an intracellular domain for signal transduction.

The extracellular domain of a CAR of the present disclosure comprises an anti-glycoMUC1 antibody or antigen binding fragment sequence (eg, as described in Section 5.1 or Embodiments 1-90).

Exemplary transmembrane and intracellular domain sequences are described in Sections 5.4.1 and 5.4.2, respectively.

A number of the fusion proteins described herein (eg, embodiments 92 and 94-96) are CARs, and the disclosures related to CARs apply to such fusion proteins.

5.4.1. Transmembrane Domains With respect to transmembrane domains, CARs can be designed to include a transmembrane domain operably linked (eg, fused) to the extracellular domain of the CAR.

Transmembrane domains can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Transmembrane regions of particular use in this disclosure include T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, It may be derived from the alpha, beta or zeta chain of CD154 (ie, it may include at least the transmembrane region thereof). In some cases, various human hinges can be employed as well, such as the human Ig (immunoglobulin) hinge.

In one embodiment, the transmembrane domain is synthetic (ie, not naturally occurring). Examples of synthetic transmembrane domains are peptides containing primarily hydrophobic residues such as leucine and valine. Preferably, a phenylalanine, tryptophan and valine triplet is expected to be found at each end of the synthetic transmembrane domain. Optionally, a short oligo or polypeptide linker, preferably 2 to 10 amino acids in length, can form a link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. The glycine-serine dyad provides a particularly suitable linker.

In one embodiment, the transmembrane domain in a CAR of the present disclosure is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence YLHLGALGRDLWGPSPVTGYHPLL.

In one embodiment, the transmembrane domain in a CAR of the present disclosure is a CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV.

In some cases, the transmembrane domain of a CAR of the present disclosure comprises a CD8a hinge domain. In one embodiment, the CD8a hinge domain comprises the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGGAVHTRGLDFAC.

5.4.2. Intracellular Domain The intracellular signaling domain of the disclosed CARs is involved in the activation of at least one normal effector function of the immune cell in which the CAR is expressed. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be a cytolytic activity or a helper activity, including secretion of cytokines. Accordingly, the term "intracellular signaling domain" refers to the portion of a protein that transduces effector function signals and directs the cell to perform specialized functions. Generally, the entire intracellular signaling domain can be employed, but in many cases it is not necessary to use the entire chain. To the extent that truncated portions of intracellular signaling domains are used, such truncated portions may be used in place of the intact chain as long as it transduces effector function signals. can. The term intracellular signaling domain is therefore meant to include any truncated portion of an intracellular signaling domain sufficient to transduce an effector function signal.

Preferred examples of intracellular signaling domains for use in the CARs of the present disclosure include T-cell receptors (TCRs) and co-receptors that act cooperatively to initiate signal transduction after binding to antigen receptors. Includes cytoplasmic sequences, as well as any derivatives or variants of these sequences, and any synthetic sequences with the same functional capacity.

Signals generated by the TCR alone may be insufficient for complete activation of T cells; secondary or costimulatory signals are also required. T cell activation is therefore dependent on two distinct classes of cytoplasmic signaling sequences: the signaling sequences that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences); It can be said to be mediated by signal transduction sequences (secondary cytoplasmic signal transduction sequences) that act in an antigen-independent manner to provide secondary or costimulatory signals.

Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex, either through stimulation or inhibition. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.

Examples of primary cytoplasmic signaling sequences containing ITAMs that have particular use in the CARs of the present disclosure include TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b. , and those derived from CD66d. It is particularly preferred that the cytoplasmic signaling molecule in the CAR of the present disclosure comprises a CD3-zeta derived cytoplasmic signaling sequence.

In a preferred embodiment, the intracytoplasmic domain of the CAR comprises the ITAM-containing primary intracytoplasmic signaling sequence domain itself (e.g., that of CD3-zeta), or any other desirable intracytoplasmic domain useful in the context of the CAR of the present disclosure. Designed to contain in combination with an intracytoplasmic domain. For example, the intracytoplasmic domain of CAR may include a CD3 zeta chain portion and a costimulatory signaling region.

Co-stimulatory signaling region refers to the portion of the CAR that contains the intracellular domain of co-stimulatory molecules. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for the efficient response of lymphocytes to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT , NKG2C, B7-H3, and a ligand that specifically binds to CD83.

The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CARs of the present disclosure may be linked to each other in a random or specified order. Optionally, short oligo or polypeptide linkers, preferably 2 to 10 amino acids in length, can form the linkage. The glycine-serine dyad provides a particularly suitable linker.

In one embodiment, the cytoplasmic domain comprises a CD3-zeta signaling domain and a CD28 signaling domain. In another embodiment, the cytoplasmic domain comprises a CD3-zeta signaling domain and a 4-1BB signaling domain.

5.5 Nucleic Acids, Recombinant Vectors and Host Cells The present disclosure provides nucleic acid molecules encoding the immunoglobulin light chain and heavy chain genes of anti-glycoMUC1 antibodies, vectors containing such nucleic acids, and the anti-glycoMUC1 antibodies of the present disclosure. Includes host cells capable of producing MUC1 antibodies. In certain embodiments, the nucleic acid molecules include anti-glycoMUC1 antibodies and antigen-binding fragments of the present disclosure (e.g., as described in Section 5.1 and Embodiments 1-90), as well as fusion proteins containing them ( e.g., as described in embodiments 91-96) and chimeric antigen receptors (e.g., as described in Section 5.4 and embodiments 97-98), and the host cell is capable of expressing them. is possible. Exemplary vectors of the disclosure are described in embodiments 111-113, and exemplary host cells are described in embodiments 114-117.

Anti-glycoMUC1 antibodies of the present disclosure can be prepared by recombinant expression of immunoglobulin light chain and heavy chain genes in host cells. To recombinantly express antibodies, host cells are transfected with one or more recombinant expression vectors carrying DNA fragments encoding the antibody's immunoglobulin light and heavy chains, thereby The chains are expressed in the host cell, and optionally they are secreted into the medium in which the host cell is cultured, from which the antibody can be recovered. Standard recombinant DNA techniques involve obtaining antibody heavy and light chain genes, incorporating these genes into recombinant expression vectors, and inserting the vectors into host cells, e.g., by Molecular Cloning; A Laboratory Manual, Second Edition ( Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, N. Y., 1989), Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds., Greene Publishing Associates, 1989) and U.S. Pat. No. 4,816,397. Used to introduce what is described in the specification.

To generate a nucleic acid encoding such an anti-glycoMUC1 antibody, first, DNA fragments encoding the light chain and heavy chain variable regions are obtained. These DNAs can be obtained by amplifying and modifying germline DNA or cDNA encoding the light and heavy chain variable sequences using, for example, polymerase chain reaction (PCR). Germline DNA sequences for human heavy and light chain variable region genes are known in the art (see, e.g., the "VBASE" human germline sequence database; Kabat et al., 1991, Sequences of Proteins). of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242; Tomlinson et al., 1992, J. Mol. Biol. 22T:116-198; and Cox et al., 1994, See also Eur. J. Immunol. 24:827-836; the contents of each of these are incorporated herein by reference).

Once DNA fragments encoding the V _H and V _L segments associated with the anti-glyco-MUC1 antibody are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques to, for example, convert variable region genes into full-length antibodies. It can be converted into a chain gene, Fab fragment gene or scFv gene. In these manipulations, a DNA fragment encoding a V _H or V _L is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" when used in this context is intended to mean that the two DNA fragments come together such that the amino acid sequences encoded by the two DNA fragments remain in frame. be done.

The isolated DNA encoding the V _H region combines the V _H encoding DNA into another DNA molecule encoding the heavy chain constant region (CH ₁ , CH ₂ , CH ₃ , and optionally CH ₄ ). By operably linking, it can be converted into a full-length heavy chain gene. The sequence of the human heavy chain constant region gene is known in the art (e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). ), DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA, IgE, IgM or IgD constant region, but in certain embodiments it is an IgG ₁ or IgG ₄ constant region. be. In the case of a Fab fragment heavy chain gene, the DNA encoding the V _H may be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

The isolated DNA encoding the V _L region can be obtained by operably linking the V _L encoding DNA to another DNA molecule encoding the light chain constant region, CL. Fab light chain gene). The sequence of the human light chain constant region gene is known in the art (e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). ), DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region, but in certain embodiments is a kappa constant region.

To create scFv genes, DNA fragments encoding V _H and V _L are prepared such that the V _H and V _L sequences can be expressed as a contiguous single-chain protein in which the V _H and V _L regions are joined by a flexible linker. may be operably linked to another fragment encoding a flexible linker, such as another fragment encoding the amino acid sequence (Gly ₄ -Ser) ₃ (e.g., Bird et al., 1988, Science 242 :423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature 348:552-554).

In order to express the anti-glyco-MUC1 antibody of the present disclosure, the DNA encoding the partial or full-length light chain and heavy chain obtained as described above is prepared such that the genes are operably linked to transcriptional and translational control sequences. It is inserted into an expression vector as shown in FIG. In this context, the term "operably linked" means that the antibody genes are ligated into the vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating transcription and translation of the antibody gene. is intended to mean. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene may be inserted into separate vectors, or, more typically, both genes are inserted into the same expression vector.

The antibody genes are inserted into the expression vector by standard methods (eg, complementary restriction site ligation on the antibody gene fragment and the vector, or blunt-end ligation if no restriction sites are present). Prior to insertion of anti-glycoMUC1 antibody-related light chain or heavy chain sequences, the expression vector may already contain antibody constant region sequences. For example, one approach to converting the V _H and V _L sequences associated with an anti-glyco-MUC1 monoclonal antibody into a full-length antibody gene is to operably link the V _H segment to a CH segment within the vector, and the V _L segment to the vector. and inserting them into an expression vector pre-encoding the heavy and light chain constant regions, respectively, so as to be operably linked to the CL segment within the CL segment. Additionally, or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chains from the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide is linked in frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide derived from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expression vectors of the present disclosure have regulatory sequences that control expression of the antibody chain genes in the host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif., 1990. Those skilled in the art will appreciate that the design of the expression vector, including the choice of regulatory sequences, may depend on such factors as the choice of host cell to be transformed, the level of protein expression desired, etc. It is expected that there will be. Suitable regulatory sequences for mammalian host cell expression include viral elements that direct high level protein expression in mammalian cells, such as promoters and/or enhancers from cytomegalovirus (CMV) (e.g., CMV promoter/enhancer), simian Promoters and/or enhancers derived from virus 40 (SV40) (e.g. SV40 promoter/enhancer), promoters and/or enhancers derived from adenoviruses (e.g. adenovirus major late promoter (AdMLP)), and promoters and/or enhancers derived from polyomas. Examples include enhancers. For further description of viral regulatory elements and their sequences, see, for example, U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al., and U.S. Pat. , 968,615.

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the present disclosure have additional sequences such as sequences that control the replication of the vector in a host cell (e.g., an origin of replication) and selectable marker genes. You can leave it there. Selectable marker genes facilitate the selection of host cells into which the vector has been introduced (e.g., U.S. Pat. Nos. 4,399,216; 4,634,665; 5,179,017). For example, the selectable marker gene typically confers resistance to drugs such as G418, hygromycin or methotrexate to a host cell into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (DHFR ^- for use with methotrexate selection/amplification in host cells) and the neo gene (for G418 selection). For expression of light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. Various forms of the term "transfection" are commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as, for example, electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection. It is intended to encompass a variety of techniques that may be used.

It is contemplated that antibodies of the present disclosure may be expressed in either prokaryotic or eukaryotic host cells. In certain embodiments, expression of antibodies, or optimal secretion of properly folded immunologically active antibodies, is performed in eukaryotic cells, such as mammalian host cells. Exemplary mammalian host cells for expressing recombinant antibodies of the present disclosure include Chinese hamster ovary (CHO cells) (e.g., Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220 DHFR ⁻ CHO cells, which are used with a DHFR selectable marker as described in, e.g., Kaufman and Sharp, 1982, Mol. Biol. 159:601-621), NSO bone marrow tumor cells, COS cells and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is in a state sufficient to permit expression of the antibody in the host cell or secretion of the antibody into the culture medium in which the host cell is grown. It is produced by culturing host cells for an extended period of time. Antibodies can be recovered from culture media using standard protein purification methods. Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. It is understood that variations on the above procedure are within the scope of the present disclosure. For example, it may be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an anti-glycoMUC1 antibody of the present disclosure.

For expression of a CAR of the present disclosure, preferably the host cell is a T cell, preferably a human T cell, as described, for example, in Section 5.4 and further in embodiments 97 and 98. In some embodiments, host cells exhibit anti-tumor immunity when the cells are cross-linked with MUC1 on tumor cells. Detailed methods for producing T cells of the present disclosure are described in Section 5.5.1.

Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both light and heavy chains that is not necessarily required for binding to glycoMUC1. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the present disclosure.

For recombinant expression of the anti-glycoMUC1 antibodies of the present disclosure, a host cell carries two expression vectors of the present disclosure, a first vector encoding a polypeptide derived from the heavy chain and a polypeptide derived from the light chain. A second vector encoding the vector may be co-transfected. The two vectors may contain the same selectable marker, or they may each contain different selectable markers. Alternatively, a single vector encoding both heavy and light chain polypeptides may be used.

Once the nucleic acid encodes one or more portions of an anti-glycoMUC1 antibody, for example, it encodes an antibody with different CDR sequences, an antibody with reduced affinity for Fc receptors, or an antibody of a different subclass. Further changes or mutations can be introduced into the coding sequence to produce a nucleic acid.

Anti-glycoMUC1 antibodies of the present disclosure can also be produced by chemical synthesis (eg, by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.). Variant antibodies can also be generated using cell-free platforms (e.g. Chu et al., Biochemia No.2, 2001 (Roche Molecular Biologicals) and Murray et al., 2013, Current Opinion in Chemical Biology, 17 :420-426).

Once the anti-glycoMUC1 antibodies of the present disclosure have been produced by recombinant expression, they can be purified by any method known in the art for the purification of immunoglobulin molecules, such as by chromatography (e.g., ion exchange, affinity, and sizing column chromatography). It can be purified by , centrifugation, fractional lysis, or by any other standard protein purification technique. Additionally, the anti-glyco-MUC1 antibodies and/or binding fragments of the present disclosure may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification. be able to.

Once isolated, anti-glycoMUC1 antibodies are optionally purified, for example by high performance liquid chromatography (see Fisher, Laboratory Techniques In Biochemistry And Molecular Biology, Work and Burdon, eds., Elsevier, 1980) or by Superdex ( Further purification may be carried out by gel filtration chromatography on a 75 column (Pharmacia Biotech AB, Uppsala, Sweden).

5.5.1. Recombinant Production of CARs in T Cells In some embodiments, nucleic acids encoding anti-glycoMUC1 CARs of the present disclosure are delivered to cells using retroviral or lentiviral vectors. Retroviral and lentiviral vectors expressing CAR can be produced using transduced cells as carriers or using cell-free local or systemic delivery of encapsulated, conjugated, or naked vectors. It can be delivered to different types of eukaryotic cells and even to tissues and whole organisms. The method used can be for any purpose for which stable expression is necessary or sufficient.

In other embodiments, CAR sequences are delivered to cells using in vitro transcribed mRNA. In vitro transcribed mRNA CARs can be expressed in different types using transfected cells as carriers or using cell-free local or systemic delivery of encapsulated, conjugated, or naked mRNA. It can be delivered to eukaryotic cells and even to tissues and whole organisms. The method used may be for any purpose for which transient expression is necessary or sufficient.

In another embodiment, a desired CAR can be expressed in a cell by a transposon.

One advantage of the RNA transfection methods of the present disclosure is that RNA transfection is inherently transient and vector-free, i.e., the RNA transgene is delivered to lymphocytes and After cell activation in vitro, it is expressed there as a minimal expression cassette without the need for any additional viral sequences. Under these conditions, integration of the transgene into the host cell genome is unlikely to occur. Cloning of cells does not necessarily depend on the transfection efficiency of the RNA or its ability to uniformly modify the entire lymphocyte population.

Genetic modification of T cells with in vitro transcribed RNA (IVT-RNA) utilizes two different strategies, both of which are continually being tested in various animal models. Cells are transfected with in vitro transcribed RNA by lipofection or electroporation. Preferably, it is desirable to use various modifications to stabilize the IVT-RNA in order to achieve sustained expression of the transferred IVT-RNA.

It is known in the literature that some IVT vectors have been utilized in a standardized manner as templates for in vitro transcription and have been genetically modified to produce stabilized RNA transcripts. . Currently, protocols used in the art include the following structure: a 5' RNA polymerase promoter that allows RNA transcription, followed by either 3' and/or 5' flanking the untranslated region (UTR). based on a plasmid vector with the gene of interest and a 3' polyadenyl cassette containing 50-70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized with a type II restriction enzyme (recognition sequence corresponds to the cleavage site) downstream of the polyadenyl cassette. The polyadenylic cassette therefore corresponds to the late poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization, extending or masking the poly(A) sequence at the 3' end. It is unclear whether this non-physiological overhang affects the amount of protein produced intracellularly from such constructs.

RNA has a number of advantages over more traditional plasmid or viral approaches. Gene expression from RNA sources does not require transcription and protein products are produced immediately after transfection. Furthermore, because the RNA only needs to enter the cytoplasm and not the nucleus, typical transfection methods result in extremely high transfection rates. In addition, plasmid-based approaches require activation of a promoter that drives expression of the gene of interest in the cells under study.

In another embodiment, RNA constructs can be delivered to cells by electroporation. For example, US Patent Application Publication No. 2004/0014645, US Patent Application Publication No. 2005/0052630, US Patent Application Publication No. 2005/0070841, US Patent Application Publication No. 2004/0059285, See formulations and techniques for electroporation of nucleic acid constructs into mammalian cells, as taught in US Patent Application Publication No. 2004/0092907. The various parameters such as electric field strength required for electroporation of any known cell type are generally known in the relevant research literature as well as numerous patents and applications in this field. See, eg, US Patent No. 6,678,556, US Patent No. 7,171,264, and US Patent No. 7,173,116. Devices for therapeutic applications of electroporation are commercially available, such as the MedPulser™ DNA Electroporation Therapy System (Inovio/Genetronics, San Diego, Calif.) and described in U.S. Pat. No. 694; U.S. Patent No. 6,516,223, U.S. Patent No. 5,993,434, U.S. Patent No. 6,181,964, U.S. Patent No. 6,241,701 and electroporation in vitro, as described in U.S. Pat. No. 6,233,482, and patents such as U.S. Pat. can also be used for transfection. Electroporation can also be used to deliver nucleic acids to cells in vitro. Accordingly, electroporation-mediated administration of nucleic acids containing expression constructs to cells, utilizing any of the many available devices and electroporation systems known to those skilled in the art, to deliver RNA of interest to target cells. provide an exciting new means of

5.5.1.1 Source of T Cells A source of T cells is obtained from a subject prior to expansion and genetic modification. The term "subject" is intended to include organisms capable of eliciting an immune response (eg, mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably the subject is a human.

T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusions, splenic tissue, and tumors. In certain embodiments of the present disclosure, various T cell lines available in the art can be used. In certain embodiments of the present disclosure, T cells can be obtained from blood units collected from a subject using various techniques known to those skilled in the art, such as Ficoll™ separation. In one preferred embodiment, cells from an individual's circulating blood are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and placed in a suitable buffer or medium for subsequent processing steps. In one embodiment of the present disclosure, cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the cleaning solution may be devoid of calcium, devoid of magnesium, or devoid of even partial divalent cations. Again, surprisingly, the initial activation step in the absence of calcium results in further activation. As one skilled in the art would readily understand, the washing steps can be performed by methods known to those skilled in the art, such as in a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell) according to the manufacturer's instructions. This may be achieved by using a Baxter CytoMate or Haemonetics Cell Saver 5). After washing, cells may be resuspended in various biocompatible buffers, such as Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, the apheresis sample may be cleaned of unwanted components and the cells resuspended directly in culture medium.

In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes by, for example, centrifugation through a PERCOLL™ gradient or countercurrent centrifugal elution. Specific subpopulations of T cells, such as CD3 ⁺ , CD28', CD4 ⁺ , CD8 ⁺ , CD45RA ⁺ and CD45RO ⁺ T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are treated with anti-CD3/anti-CD28 (i.e., 3 x 28) conjugated beads, such as DYNABEADS® M-450 CD3/CD28T, for positive selection of desired T cells. Isolated by incubation for a sufficient period of time. In one embodiment, the period is about 30 minutes. In further embodiments, the time period ranges from 30 minutes to 36 hours or more, and all integer values therebetween. In further embodiments, the period of time is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the time period is 10 to 24 hours. In one preferred embodiment, the incubation period is 24 hours. In the case of isolation of T cells from patients with leukemia, the use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times allow isolation of T cells in any situation where T cells are extremely scarce compared to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissues or immunocompromised individuals. It can be used to. Furthermore, the use of longer incubation times can increase the capture efficiency of CD8+ T cells. Thus, by simply shortening or lengthening the time that T cells are bound to CD3/CD28 beads and/or by increasing or decreasing the bead to T cell ratio (as described further herein). Subpopulations of T cells can be preferentially selected for presence or absence, at the beginning of the culture, or at other time points during the process. In addition, by increasing or decreasing the proportion of anti-CD3 and/or anti-CD28 antibodies on beads or other surfaces, subpopulations of T cells can be made present at the beginning of the culture or at other desired time points. You can choose it with priority regardless of whether you do it or not. It is expected that those skilled in the art will recognize that multiple selections can also be used in the context of this disclosure. In certain embodiments, it may be desirable to perform a selection procedure and use "unselected" cells in the activation and expansion process. "Unselected" cells can also be subjected to further rounds of selection.

Enrichment of T cell populations by negative selection can be achieved in conjunction with antibodies directed against surface markers specific to negatively selected cells. One method is cell sorting and/or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers displayed on negatively selected cells. For example, to enrich for CD4 ⁺ cells by negative selection, monoclonal antibody cocktails typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich for or positively select regulatory T cells, which typically express CD4 ⁺ , CD25 ⁺ , CD62L ^hi , GITR ⁺ , and FoxP3 ⁺ . Alternatively, in certain embodiments, regulatory T cells are depleted by anti-C25 conjugated beads or other similar selection methods.

For isolation of desired cell populations by positive or negative selection, the concentrations of cells and surfaces (eg, particles such as beads) may vary. In certain embodiments, it may be desirable to significantly reduce the volume in which beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells per ml is used. In one embodiment, a concentration of 1 billion cells per ml is used. In further embodiments, more than 100 million cells per ml are used. In further embodiments, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells per ml are used. In yet other embodiments, cell concentrations from 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells per ml are used. In further embodiments, concentrations of 125 million or 150 million cells per ml may be used. The use of high concentrations can result in increased cell yield, cell activation, and cell expansion. Additionally, the use of high cell concentrations may result in more efficient capture of cells that may have weak expression of the target antigen of interest, e.g. CD28-negative T cells, or in samples where many tumor cells are present (i.e., leukemia blood , tumor tissue, etc.). Such cell populations are expected to have potential therapeutic value and would be desirable to obtain. For example, the use of high concentrations of cells allows for more efficient selection of CD8 ⁺ T cells, which usually have relatively weak CD28 expression.

In related embodiments, it may be desirable to use lower cell concentrations. By significantly diluting the mixture of T cells and surfaces (eg, particles such as beads), interactions between particles and cells are minimized. This selects cells that express large amounts of the desired antigen to be bound to the particles. For example, CD4 ⁺ T cells express higher levels of CD28 and are captured more efficiently than CD8 ⁺ T cells at dilute concentrations. In one embodiment, the cell concentration used is 5×10 ⁶ /ml. In other embodiments, the concentration used may range from about 1 x 10 ⁵ /ml to 1 x 10 ⁶ /ml, and any integer value therebetween.

In other embodiments, cells may be incubated on a rotator for varying lengths of time and at varying speeds, either at 2°C to 10°C or at room temperature.

T cells for stimulation may also be frozen after the washing step. Without wishing to be bound by theory, the freezing and subsequent thawing step provides a more homogeneous product by removing granulocytes and some monocytes in the cell population. After a washing step to remove plasma and platelets, the cells may be suspended in a freezing solution. Although many freezing solutions and parameters are known in the art and would be expected to be useful in this situation, one method is PBS containing 20% DMSO and 8% human serum albumin, or 10% dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20 % human serum albumin, and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A, and then the cells were incubated at a rate of 1°/min. The sample is frozen at −80° C. and stored in the gas phase in a liquid nitrogen storage tank. Other controlled freezing methods can also be used, as well as immediate freezing at -20°C or uncontrolled freezing in liquid nitrogen.

In certain embodiments, prior to activation using the methods of the present disclosure, cryopreserved cells are thawed, washed as described herein, and left undisturbed at room temperature for 1 hour. .

It is also contemplated in the context of this disclosure to collect blood samples or apheresis products from the subject at a time prior to when the increased cells described herein are deemed necessary. Accordingly, the source of cells to be expanded can be harvested at any time point required and the desired cells, e.g. can be isolated and frozen for later use in T cell therapy for diseases or conditions described in . In one embodiment, the blood sample or apheresis is taken from a generally healthy subject. In certain embodiments, the blood sample or apheresis is taken from a generally healthy subject who is at risk of developing the disease but has not yet developed the disease, and the cells of interest are isolated for later use. and frozen. In certain embodiments, T cells can be expanded, frozen, and used at a later time. In certain embodiments, samples are collected from patients immediately after diagnosis of a particular disease described herein, but prior to any treatment. In further embodiments, various related treatment modalities such as, but not limited to, natalizumab, efalizumab, antiviral agents, chemotherapeutic agents, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate, and before treatment with agents such as FK506, antibodies, or other immunodepleting agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiation. Cells are isolated from a blood sample or apheresis from a subject. These drugs either inhibit calcineurin, a calcium-dependent phosphatase (cyclosporine and FK506), or inhibit p70S6 kinase, which is important for growth factor-induced signal transduction (rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). In further embodiments, the cells are administered in combination with (e.g., prior to) bone marrow or stem cell transplantation or T cell ablation therapy using any of the chemotherapeutic agents, e.g., fludarabine, external radiation therapy (XRT), cyclophosphamide. isolated and frozen for each patient for subsequent use (, concurrently, or subsequently).

In a further embodiment of the present disclosure, T cells are obtained from the patient immediately after treatment. In this regard, after certain cancer treatments, especially after treatment with drugs that damage the immune system, the quality of the T cells obtained immediately after the treatment during the period when the patient would normally be expected to recover from the treatment It has been observed that the drug may be optimal or improved with respect to its ex vivo increasing ability. Similarly, after ex vivo manipulation using the methods described herein, these cells may be in a favorable situation for enhanced engraftment and in vivo expansion. Therefore, in the context of the present disclosure, it is anticipated that blood cells, including T cells, dendritic cells, or cells of other hematopoietic lineages, will be collected during this recovery period. Additionally, in certain embodiments, the mobilization (e.g., mobilization with GM-CSF) and conditioning regimens specifically promote the recolonization, recirculation, regeneration, and/or regeneration of specific cell types during a defined time frame following therapy. or can be used to create a condition in a subject that is favorable for increase. Exemplary cell types include T cells, B cells, dendritic cells, and other immune system cells.

5.5.1.2 Activation and Expansion of T Cells T cells are generally treated in a variety of ways, e.g., U.S. Patent No. 6,352,694; , 680 specification; 6,692,964 specification; 5,858,358 specification; 6,887,466 specification; 6,905,681 specification; 7,144 , 575 specification; 7,067,318 specification; 7,172,869 specification; 7,232,566 specification; 7,175,843 specification; 5,883 , 223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Publication No. 20060121005. activated and increased using such methods.

Generally, T cells of the present disclosure are expanded by contacting them with a surface attached with an agent that stimulates CD3/TCR complex associated signals and a ligand that stimulates costimulatory molecules on the T cell surface. In particular, T cell populations can be isolated, for example, by contacting with an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or together with a calcium ionophore, a protein kinase C activator (e.g., bryostatin). can be stimulated as described herein. For co-stimulation of accessory molecules on the surface of T cells, ligands that bind accessory molecules are used. For example, a population of T cells can be contacted with anti-CD3 and anti-CD28 antibodies under conditions appropriate to stimulate T cell proliferation. Anti-CD3 and anti-CD28 antibodies to stimulate proliferation of either CD4 ⁺ T cells or CD8 ⁺ T cells. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), which can generally be used as well as other methods known in the art (Berg et al. ., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1- 2):53-63, 1999).

In certain embodiments, primary and costimulatory signals for T cells can be provided by a variety of protocols. For example, each signal-providing agent may be in solution or coupled to a surface. When coupled to a surface, the agent may be coupled to the same surface (ie, in a "cis" configuration) or to another surface (ie, in a "trans" configuration). Alternatively, one drug may be coupled to the surface and the other drug in solution form. In one embodiment, the agent that provides the costimulatory signal is bound to the cell surface and the agent that provides the primary activation signal is in solution or coupled to the surface. In certain embodiments, both agents may be in the form of a solution. In another embodiment, the drug may be in soluble form and then cross-linked to a surface, such as a cell expressing an Fc receptor, or to an antibody or other binding agent that is expected to bind to the drug. Good too. In this regard, see, for example, U.S. Pat. Please refer.

In one embodiment, the two agents are immobilized on beads, either on the same bead, ie, "cis," or on separate beads, ie, "trans." As an example, the agent that provides the primary activation signal is an anti-CD3 antibody or antigen-binding fragment thereof, and the agent that provides the costimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof, and both agents are equivalent The same beads are immobilized together in an amount of molecules. In one embodiment, a 1:1 ratio of each antibody conjugated to beads is used for CD4 ⁺ T cell expansion and T cell proliferation. In certain embodiments of the present disclosure, the ratio of anti-CD3:CD28 antibodies bound to beads is such that an increase in T cell expansion is observed compared to the increase observed using a 1:1 ratio. is used. In one particular embodiment, an increase of about 1 to about 3 times is observed compared to the increase observed using a 1:1 ratio. In one embodiment, the ratio of CD3:CD28 antibodies bound to beads ranges from 100:1 to 1:100, and all integer values therebetween. In one aspect of the present disclosure, more anti-CD28 antibodies than anti-CD3 antibodies are bound to the particles, ie, the CD3:CD28 ratio is less than 1. In certain embodiments of the present disclosure, the ratio of anti-CD28 antibodies to anti-CD3 antibodies bound to beads is greater than 2:1. In one particular embodiment, antibodies coupled to beads at a CD3:CD28 ratio of 1:100 are used. In another embodiment, antibodies coupled to beads with a CD3:CD28 ratio of 1:75 are used. In a further embodiment, antibodies coupled to beads at a CD3:CD28 ratio of 1:50 are used. In another embodiment, antibodies coupled to beads at a CD3:CD28 ratio of 1:30 are used. In one preferred embodiment, antibodies coupled to beads at a CD3:CD28 ratio of 1:10 are used. In another embodiment, antibodies coupled to beads at a CD3:CD28 ratio of 1:3 are used. In yet another embodiment, antibodies coupled to beads with a CD3:CD28 ratio of 3:1 are used.

Particle to cell ratios of 1:500 to 500:1, and any integer value therebetween, can be used to stimulate T cells or other target cells. As one skilled in the art will readily understand, the particle to cell ratio may depend on the particle size relative to the target cells. For example, small sized beads can bind only a few cells, whereas larger beads can bind many cells. In certain embodiments, the ratio of cells to particles ranges from 1:100 to 100:1, and any integer value therebetween, and in further embodiments, the ratio ranges from 1:9 to 9:1, and Including any integer value in between, these can also be used to stimulate T cells. The ratio of anti-CD3 and anti-CD28 coupled particles to T cells at which T cell stimulation occurs may vary as described above, but certain preferred values include 1:100, 1:50, 1 :40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1 , 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1, one preferred ratio being , a particle to T cell ratio of at least 1:1. In one embodiment, a particle to cell ratio of 1:1 or lower is used. In one particular embodiment, the preferred particle:cell ratio is 1:5. In further embodiments, the ratio of particles to cells can be varied depending on the day of stimulation. For example, in one embodiment, the ratio of particles to cells is 1:1 to 10:1 on day 1, and then, up to day 10, additional particles are added to a final ratio of 1:1 to 1:10. The ratio is added to cells daily or every other day (based on cell number on the day of addition). In one particular embodiment, the ratio of particles to cells is 1:1 on the first day of stimulation and adjusted to 1:5 on the third and fifth days of stimulation. In another embodiment, particles are added daily or every other day to a final ratio of 1:1 on day 1 and 1:5 on days 3 and 5 of stimulation. In another embodiment, the ratio of particles to cells is 2:1 on day 1 of stimulation and adjusted to 1:10 on days 3 and 5 of stimulation. In another embodiment, particles are added daily or every other day to a final ratio of 1:1 on day 1 and 1:10 on days 3 and 5 of stimulation. It is anticipated that those skilled in the art will appreciate that various other ratios may be suitable for use in the present disclosure. In particular, the ratio is expected to vary depending on particle size and cell size and type.

In a further embodiment of the present disclosure, cells, such as T cells, are combined with drug-coated beads, after which the beads and cells are separated and the cells are then cultured. In an alternative embodiment, the drug-coated beads and cells are not separated but are cultured together prior to culturing. In a further embodiment, beads and cells are first concentrated by application of force, such as magnetic force, resulting in increased ligation of cell surface markers, thereby inducing stimulation of the cells.

As an example, cell surface proteins can be ligated by contacting T cells with paramagnetic beads (3 x 28 beads) attached with anti-CD3 and anti-CD28. In one embodiment, cells (e.g., ¹⁰ to ¹⁰ T cells) and beads (e.g., DYNABEADS® M-450CD3/CD28T paramagnetic beads in a 1:1 ratio) are grown in a buffer. , preferably in PBS (free of divalent cations such as calcium and magnesium). Again, one skilled in the art can readily understand that any cell concentration can be used. For example, target cells may be very rare in a sample, may only make up 0.01% of the sample, or the entire sample (i.e., 100%) may be made up of target cells of interest. You can. Therefore, any number of cells is within the scope of the present disclosure. In certain embodiments, it is desirable to significantly reduce the volume in which particles and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum cell-to-particle contact. There is. For example, in one embodiment, a concentration of about 2 billion cells per ml is used. In another embodiment, more than 100 million cells per ml are used. In further embodiments, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells per ml are used. be done. In yet other embodiments, cell concentrations of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells per ml are used. In further embodiments, concentrations of 125 million or 150 million cells per ml may be used. The use of high concentrations can result in increased cell yield, cell activation, and cell expansion. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may weakly express the target antigen of interest, such as CD28-negative T cells. It is anticipated that such cell populations may have therapeutic value and may be desirable to obtain in certain embodiments. For example, the use of high concentrations of cells allows for more efficient selection of CD8+ T cells, which usually have relatively weak CD28 expression.

In one embodiment of the present disclosure, the mixture can be cultured for a period of several hours (about 3 hours) to about 14 days, or any integer value therebetween. In another embodiment, the mixture can be cultured for 21 days. In one embodiment of the present disclosure, beads and T cells are cultured together for about 8 days. In another embodiment, beads and T cells are cultured together for 2-3 days. Several cycles of stimulation may be desirable so that the T cells can be cultured for 60 days or longer. Suitable conditions for T cell culture include serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL. -10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives for cell proliferation known to those skilled in the art. media (eg, Minimum Essential Medium or RPMI Medium 1640 or X-vivo15 (Lonza)). Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. The medium may be serum-free or supplemented with amino acids, sodium pyruvate, and vitamins, or an appropriate amount of serum (or plasma) or a defined set of hormones, and/or sufficient for T-cell proliferation and expansion. RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, any of which were supplemented with an appropriate amount of cytokines. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in the culture of cells that are to be injected into the subject. Target cells are maintained under conditions necessary to support proliferation, eg, at a suitable temperature (eg, 37° C.) and atmosphere (eg, air plus 5% CO ₂ ).

T cells exposed to different stimulation times may exhibit different characteristics. For example, a typical blood or peripheral blood mononuclear cell product that has undergone apheresis has a larger helper T cell population (T _H , CD4 ⁺ ) than the cytotoxic or suppressor T cell population (T _C , CD8 ⁺ ). . Expansion of T cells by stimulating CD3 and CD28 receptors ex vivo produces a population of T cells consisting primarily of T _H cells before about day 8-9, but before about day 8-9. Afterwards, the population of T cells includes a much larger population of _TC cells. Therefore, depending on the goal of treatment, it may be advantageous to infuse a subject with a T cell population that primarily comprises T _H cells. Similarly, if an antigen-specific subset of _TC cells has been isolated, it may be beneficial to expand this subset to a higher degree.

Furthermore, in addition to the CD4 and CD8 markers, other phenotypic markers change significantly, but largely reproducibly, during the course of the pleocytosis process. Such reproducibility thus enables the ability to tailor activated T cell products to specific purposes.

5.6 Compositions The anti-glycoMUC1 antibodies and/or anti-glycoMUC1 ADCs of the present disclosure may be prepared in compositions comprising the anti-glycoMUC1 antibodies and/or ADCs and one or more carriers, excipients and/or diluents. It may be in the form of The composition may be formulated for a particular use, eg, veterinary use or human pharmaceutical use. The composition (e.g., dry powder, liquid formulation, etc.) and form of excipient, diluent, and/or carrier used will depend on the intended use of the antibody and/or ADC, as well as the therapeutic use, mode of administration, etc. It is expected that this will be determined separately.

For therapeutic use, the composition may be supplied as part of a sterile pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition may be in any suitable form (depending on the desired method of administering it to the patient). Pharmaceutical compositions can be administered to a patient by a variety of routes, including oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, topical or topical. The most suitable route of administration in any given case is expected to depend on the particular antibody and/or ADC, the subject, and the nature and severity of the subject's disease and physical condition. Typically, pharmaceutical compositions will be administered intravenously or subcutaneously.

Pharmaceutical compositions can conveniently be provided in unit dosage forms containing a predetermined amount of the anti-glycoMUC1 antibodies and/or anti-glycoMUC1 ADCs of the present disclosure per dose. It is expected that the amount of antibody and/or ADC contained in a unit dose will depend on the disease being treated, as well as other factors well known in the art. Such a unit dosage may be in the form of a lyophilized dry powder containing an amount of antibody and/or ADC suitable for single administration, or it may be in liquid form. The dry powder unit dosage form may be packaged in a kit with a syringe, a suitable amount of diluent and/or other ingredients useful for administration. Unit dosages in liquid form can be conveniently presented in the form of a syringe prefilled with an amount of antibody and/or ADC suitable for single administration.

Pharmaceutical compositions may also be supplied in bulk form containing an amount of ADC suitable for multiple administration.

For storage as a lyophilized formulation or an aqueous solution, the pharmaceutical composition comprises an antibody and/or ADC of desired purity in any pharmaceutically acceptable carrier, excipient, or carrier typically employed in the art. or stabilizers (all of which are referred to herein as "carriers"), i.e., buffers, stabilizers, preservatives, tonicity agents, nonionic detergents, antioxidants, and others. may be prepared by mixing with a mixture of additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed.1980). Such excipients should be non-toxic to recipients at the dosages and concentrations employed.

Buffers help maintain pH in a range near physiological conditions. Buffers may be present at varying concentrations, but are typically expected to be present at concentrations ranging from about 2 mM to about 50 mM. Suitable buffers for use in the present disclosure include, for example, citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-citric acid monosodium mixtures, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartrate buffers (e.g., tartaric acid-tartrate mixtures, etc.) sodium mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumaric acid buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-fumaric acid) gluconate buffer (e.g., gluconate-sodium glyconate mixture, gluconate-sodium hydroxide mixture, gluconate-potassium glyuconate mixture, etc.), oxalate buffer liquids (e.g., oxalic acid-sodium oxalate mixtures, oxalic acid-sodium hydroxide mixtures, oxalic acid-potassium oxalate mixtures, etc.), lactic acid buffers (e.g., lactic acid-sodium lactate mixtures, lactic acid-sodium hydroxide mixtures, lactic acid -potassium lactate mixtures, etc.) and acetate buffers (eg, acetic acid-sodium acetate mixtures, acetic acid-sodium hydroxide mixtures, etc.) and their salts. In addition, phosphate buffers, histidine buffers and trimethylamine salts such as Tris can be used.

Preservatives may be added to prevent microbial growth and can be added in amounts ranging from about 0.2% to 1% (w/v). Preservatives suitable for use in the present disclosure include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide). chloride), hexamethonium chloride, and alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonic agents, sometimes also known as "stabilizers," can be added to ensure isotonicity of the liquid compositions of the present disclosure, examples of which include polyhydric sugar alcohols. , for example sugar alcohols having 3 or more hydroxyl groups, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Stabilizers refer to a broad category of excipients that vary in functionality from bulking agents to additives that solubilize therapeutic agents or help prevent denaturation or adhesion to container walls. It can be something. Typical stabilizers are polyhydric sugar alcohols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc. organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, etc., e.g. cyclitol, e.g. inositol; polyethylene glycol; amino acid polymers; e.g. urea, Sulfur-containing reducing agents such as glutathione, thioctic acid, sodium thioglycolate, thioglycerol, alpha-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (e.g. peptides of 10 or fewer residues) proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose, glucose; monosaccharides such as lactose, maltose, sucrose and trehalose; and trisaccharides such as raffinose; as well as polysaccharides such as dextran. The stabilizer may be present in an amount ranging from 0.5 to 10 wt% relative to the weight of the ADC.

Nonionic surfactants or detergents (also known as "wetting agents") may be added to help solubilize the glycoprotein, as well as protect the glycoprotein from agitation-induced aggregation. , thereby also allowing the formulation to be exposed to stressed shear surfaces without causing protein denaturation. Suitable nonionic surfactants include polysorbates (such as 20, 80), polyxamers (such as 184, 188), and pluronic polyols. Nonionic surfactants may be present in a range of about 0.05 mg/mL to about 1.0 mg/mL, such as about 0.07 mg/mL to about 0.2 mg/mL.

Additional hybrid excipients include bulking agents (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and cosolvents.

5.7 Methods of Use The anti-glycoMUC1 antibodies or binding fragments described herein can be used in a variety of diagnostic assays. For example, antibodies and binding fragments can be used in immunoassays, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays, such as immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS). ), and Western blotting.

The anti-glycoMUC1 antibodies or binding fragments described herein are also useful for in vivo radiographic imaging, where they are labeled with a detectable moiety such as a radiopaque agent or radioisotope. The labeled antibody is administered to a subject, preferably into the bloodstream, and the presence and location of the labeled antibody in the host is assayed. This imaging technique is useful in the staging and treatment of malignant tumors.

The anti-glycoMUC1 antibodies or binding fragments, ADCs and CARs described herein are useful for treating cancers that express glycoMUC1, particularly epithelial cancers such as breast cancer, ovarian cancer, pancreatic cancer, and lung cancer. It is useful for

When using a CAR of the present disclosure for therapy, the treatment method of the present disclosure may be administered to a subject having a tumor that expresses glycoMUC1, e.g., as described in Section 5.4 or Embodiment 97 or Embodiment 98. comprising administering an effective amount of genetically modified cells engineered to express a disclosed CAR. Methods of engineering cells, particularly T cells, to express CAR are described in Section 5.5.1.

[Example 1]
Identification of anti-glycoMUC1 antibodies 6.1.1. Summary Chemoenzymatic synthesis of multi-repeat MUC1 glycopeptides with different O-glycan densities and Tn (GalNAcα1-O-Ser/Thr) glycoforms was developed using recombinant glycosyltransferases. Different polypeptide GalNAc-transferase isoforms were used to assign O-glycan occupancy sites (Bennett et al., 1998). The optimal vaccine design was found to be a Tn glycoform with high O-glycan density, and glycopeptides conjugated to KLH were found to overcome resistance. In wild-type Balb/c mice, glycopeptides with complete O-glycan occupancy elicited the strongest antibody responses that reacted with MUC1 expressed in breast cancer cell lines, suggesting that this represents the most effective vaccine design. be. The humoral immune response elicited showed surprising specificity for cancer cells.

6.1.2. Materials and Methods 6.1.2.1 Chemoenzymatic Synthesis of Multimeric Tn MUC1 Glycopeptide The 60-mer (VTSAPDTRPAPGSTAPPAHG) n=3 (SEQ ID NO: 47) peptide of MUC1 was first reported by Fontenot et al., 1993. It was synthesized as described. The control peptides used were obtained from the tandem repeats (TR) of MUC2 (PTTTPISTTTMVTPTPTPTC) (SEQ ID NO: 51) and MUC4 (CPLPVTDTSSASTGHATPLPV) (SEQ ID NO: 52). Purified recombinant human glycosyltransferase polypeptides GalNAc-T2, GalNAc-T4, and GalNAc-T1 as described in US Pat. No. 6,465,220 (Bennett et al., 1998; Peptides were glycosylated in vitro using Schwientek et al., 2002). GalNAc glycosylation of peptides was performed in a reaction mixture (1 mg/mL peptide) containing 25 mM cacodylate buffer (pH 7.4), 10 mM MnCl2, 0.25% Triton X-100, and 2 mM UDP-GalNAc. It was executed with Glycosylation of a 60-mer peptide (MUC160Tn6) with two GalNAc per mg TR was achieved using GalNAc-T1. Incorporation of 3 GalNAc per TR (MUC160Tn9) was achieved using GalNAc-T2. Replacement of all five putative O-glycosylation sites in MUC1 TR (MUC160Tn15) was performed using MUC160Tn9 as substrate in reaction with GalNAc-T4. Glycosylation was monitored using a nanoscale reverse phase column (Poros R3, PerSeptive Biosystems, Framingham, MA) and MALDI-TOF mass spectrometry. Glycopeptides were loaded onto a Zorbax 300SB-C3 column (9.4 mm x 25 cm) (Agilent Technologies, Palo Alto, CA) by high performance liquid chromatography (HPLC). Quantification and estimation of the yield of the glycosylation reaction was performed by comparison of HPLC peaks by UV210 absorbance using 10 μg peptide weight as standard. The yield of GalNAc glycosylation of peptides was 80-90% overall recovery. Purified glycopeptides were characterized by MALDI-TOF mass spectrometry on a Voyager DE or Voyager DE Pro MALDI-TOF mass spectrometer (PerSeptive Biosystems) with delayed extraction. The MALDI matrix was 10 g/L 2,5-dihydroxybenzoic acid (Aldrich, Milwaukee, WI) dissolved in a 2:1 mixture of 0.1% TFA and 30% acetonitrile in water. Samples dissolved in 0.1% TFA to a concentration of approximately 1 pmol/uL were prepared for analysis by placing 1 μL of sample solution into the probe tip followed by 1 uL of matrix. All mass spectra were acquired in linear mode. Data processing was performed using GRAMS/386 software (Galactic Industries, Salem, NH).

6.1.2.2 Immunization Protocol Glycopeptides were coupled to KLH (Pierce, Rockford, IL) using glutaraldehyde. The efficiency of conjugation was assessed by analyzing the reaction by size exclusion chromatography on a PD-10 column using an anti-MUC1 ELISA of the fractions. Essentially all reactivity was found in the excluded fractions, with non-significant reactivity found in the non-excluded fractions predicted to contain the peptide. Further evaluation included comparative titration analysis of KLH conjugates with corresponding glycopeptides in ELISA. Near complete conjugation should result in a KLH to glycopeptide ratio of 1:300, and both analyzes showed that conjugation was near complete. Female Balb/c wild type mice were injected subcutaneously with 10 or 15 μg (glyco)peptide (1:1 mixture with Freund's adjuvant, Sigma) in a total volume of 200 uL. Mice received four immunizations 14 days apart, and blood samples were obtained by tail or eye bleed 1 week after the third and fourth immunizations.

6.1.2.3 Generation of Mouse MAb Anti-Tn-MUC1 MAbs were generated from wild-type Balb/c mice immunized with a fully GalNAc-glycosylated 60-mer MUC1 glycopeptide coupled to KLH. produced. Screening was based on glycopeptide ELISA followed by immunocytology using breast cancer cell lines (MCF7 and T47D) and immunohistology using cancer tissue. Selection was made based on similar reactivity patterns to total serum from the same mice.

6.1.2.4 ELISA
ELISA was performed using 96-well MaxiSorp plates (Nunc, Denmark). Plates were coated with 1 μg/mL glycopeptide in bicarbonate-carbonate buffer (pH 9.6) overnight at 4°C and 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). and subsequent incubation with serum (diluted in PBS) or MAb for 2 hours at room temperature. The conjugated antibodies were purified using peroxidase-conjugated rabbit anti-mouse immunoglobulin (DakoCytomation, Glostrup, Denmark) or isotype-specific antibodies peroxidase-conjugated goat anti-mouse IgM, IgG1, IgG2a, IgG2b, or IgG3 (Southern Biotechnology A). ssociates, Birmingham , AL). Plates were developed with O-phenylenediamine tablets (DakoCytomation) and read at 492 nm. Control antibodies included anti-MUCl antibodies HMFG2 and SM3 (Burchell et al., 1987) and anti-carbohydrate antibodies 5F4 (Tn) and 3F1 (STn) (Mandel et al., 1991). Control sera included mice immunized with MUC4 mucin peptide linked to KLH.

6.1.3. Results A glycopeptide-specific mAb against GalNAc-MUC1 was produced using a 60-mer glycopeptide of GalNAc-MUC1 conjugated to KLH as the immunogen. Using an ELISA assay, the generated mAb GO2 (5F7) showed that the MUC1 tandem was glycosylated in vitro using purified recombinant human glycosyltransferases GalNAc-T1, GalNAc-T2, and GalNAc-T4. It reacted specifically with the repeat (VTSAPDTRPAPGSTAPPAHG) ₃ (SEQ ID NO: 47) and did not react with the corresponding MUC1 peptide without a GalNAc-residue or an unrelated glycopeptide with the same type of Tn glycoform. Figure 1 shows the results of the ELISA assay.

[Example 2]
Characterization of anti-glycoMUC1 antibodies 6.2.1. Summary Monoclonal antibody GO2 (5F7) was characterized with respect to the specificity of its binding to the Tn glycoform of MUC1 associated with cancer cells.

6.2.2. Materials and Methods 6.2.2.1 Immunocytochemistry Cell lines were fixed for 10 minutes in ice-cold acetone or methanol:acetone. Fixed cells were incubated with mouse serum (1:200/1:400/1:800) or MAbs overnight at 5°C, followed by fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin (DakoCytomation). Incubated for 45 minutes at room temperature. Slides were mounted in glycerol containing p-phenylenediamine and examined with a Zeiss fluorescence microscope (FluoresScience, Hallbergmoos, Germany).

6.2.2.2 Immunohistochemistry A formalin-fixed paraffin wax-embedded tissue of breast cancer was obtained. All cases were conventionally classified by histology. The avidin-biotin peroxidase complex method was used for immunostaining. Paraffin sections were dewaxed, rehydrated, and treated with 0.5% _H2O2 in _methanol for 30 min. Sections were rinsed in TBS and incubated with rabbit non-immune serum for 20 minutes. Sections were rinsed and incubated with primary antibodies overnight at 5°C. Sections were rinsed and incubated for 30 min with biotin-labeled rabbit anti-mouse serum (DakoCytomation) diluted 1:200 in TBS, rinsed in TBS and incubated for 1 h with avidin-biotin peroxidase complex (DakoCyto-mation). . Sections were rinsed with TBS and developed with freshly prepared 0.05% 3,3'-diaminobenzidine tetrahydrochloride in 0.05 M TBS containing 0.1% H ₂ O ₂ . Sections were stained with hematoxylin, dehydrated, and mounted.

6.2.3. Results Immunohistochemistry of colorectal cancer, pancreatic cancer, and invasive mammary carcinoma was performed using GO2. Staining of colorectal cancer tissue (Figure 2) demonstrated strong labeling of intracellular and surface structures in the majority of cancer cells. In contrast, reactivity against healthy columnar epithelial cells was absent or very low. Labeling in healthy cells was restricted to intracellular structures. This is expected to be due to the presence of large amounts of biosynthetic intermediates (GalNAc modified glycoproteins) in cells with high secretory capacity, such as the columnar epithelium of the colon. A similar pattern was observed in pancreatic (Figure 3) and breast cancer tissue (Figure 4), showing strong reactivity with cancer cells but no reactivity with intracellular structures in surrounding healthy epithelial or connective tissue cells. There was no or only limited reactivity.

[Example 3]
Sequence analysis of anti-glycoMUC1 antibody mRNA from a hybridoma producing monoclonal antibody GO2 (5F7) was prepared, reverse transcribed, and sequenced.

The nucleotide sequences encoding the heavy and light chain variable regions along with their signal sequences are set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. The heavy and light chain variable regions encoded by SEQ ID NO: 11 and SEQ ID NO: 12 are set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The predicted mature variable region sequences (after cleavage of the signal peptide) are set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and encoded by SEQ ID NO: 13 and SEQ ID NO: 14, respectively. The predicted heavy chain CDR sequences (IMGT definitions) are set forth in SEQ ID NOs: 5-7, respectively, and the predicted light chain CDR sequences (IMGT definitions) are set forth in SEQ ID NOs: 8-10, respectively.

[Example 4]
Drug delivery to cancer cells using anti-glycoMUC1 antibody 6.4.1. Summary Monoclonal antibody GO2 (5F7) was tested for its ability to deliver cytotoxic substances to target cells.

6.4.2. Materials and Methods OVCar human ovarian cancer cells were added to 24-well cell culture plates at 1,000 cells/well. Monoclonal antibody GO2 and a secondary antibody conjugated to the anti-tubulin agent monomethyl auristatin F (MMAF) (anti-mFc-NC-MMAF) (Moradec catalog number AM-101-AF) were added to the plate and the following concentrations (in μg/ml) of GO2 and ADC were obtained.

Plates were incubated at 37°C for 48 hours. After 48 hours of incubation, AlamarBlue® (Invitrogen) was added to each well and fluorescence at 600 nm was measured.

6.4.3. Results Figure 5 shows the results. The results show that cytotoxicity is dependent on the concentration and presence of the primary antibody (GO2) as well as the concentration and presence of the secondary ADC-conjugated antibody. In other words, GO2 induces cytotoxicity in this cancer cell line when coupled with a secondary antibody with the cytotoxic agent MMAF.

[Example 5]
Quantification of circulating tumor cells using anti-Muc1 antibodies 6.5.1. Summary Monoclonal antibody GO2 (5F7) was tested for its ability to be used to quantify circulating tumor cells.

6.5.2. Materials and Methods GO2 was conjugated to magnetic separation beads and allowed to interact with samples of tumor cells at different concentrations. Cells and beads were pulled out of the solution using a magnet and washed several times to remove unbound material. GO2 conjugated to horseradish peroxidase was then applied to magnetic separation beads containing bound cancer cells, incubated, and then unbound conjugated GO2 was washed away. Colorimetric reactions were performed using TNB as the substrate. The reaction was terminated using sulfuric acid and then the OD450 reading of the sample was taken.

6.5.3. Results Figure 6 shows the results. Assay results demonstrated GO2 tumor cell binding.

[Example 6]
Immunohistochemical staining of tumor tissue using anti-glycoMUC1 antibody 6.6.1. Materials and Methods Sections from six formalin-fixed paraffin-embedded (FFPE) tissue microarrays (TMAs) were cut to 2.5 μm thickness. TMA from breast cancer (BC), colorectal cancer (CRC), ovarian cancer (OVC), non-small cell lung cancer (NSCLC) and prostate cancer (PrC) tumors were used in the study. Between 25 and 47 tumor tissue cores per TMA were available for evaluation. Core size was 1 mm, 2 mm or 3 mm depending on TMA. Each tissue core is a specimen from one patient.

Staining was performed with a Discovery XT autostainer (Ventana Medical Systems). After antigen retrieval using Cell Condition 1 (CC1) solution (Ventana Medical Systems), GO2 was applied at a concentration of 1 μg/mL in Dako green medium antibody diluent and incubated for 60 min at 37 °C. did. GO2 binding to tumor cells was detected using the Optiview DAB IHC detection kit (Ventana Medical Systems) visualized with DAB (brown precipitate).

6.6.2. Results Figure 7 shows representative images of MUC1 positive TMA tumor cores. In BC and OVC TMA, the majority of spots (>90%) showed moderate or strong binding of GO2 to tumor cells. 70% and 51% of NSCLC and CRC cases showed moderate and strong antibody binding to tumor cells, respectively. In prostate cancer, antigen expression appeared to be lower. Only 28% of the spots in TMA1 showed moderate or strong staining intensity when GO-2 was applied. The staining pattern was always intracytoplasmic and in many cases membrane bound. Apical membrane staining patterns were not observed in most cores.

[Example 7]
Production and purification of anti-MUC1 antibodies in a T cell bispecific (TCB) format 6.7.1. Materials and Methods 6.7.1.1 Production of Expression Vectors Knob-into-hole and P329G/L234A/L235A (“PGLALA”) mutations in the Fc region, and MUC1 CH1 (147E/213E; “EE”) and The GO2 antibody was converted to a TCB format containing charged residues in the CL (123R/124K; "RK") region (see SEQ ID NOs: 43-46). FIG. 8 illustrates TCB antibodies. Briefly, the variable heavy and variable light chains of the GO2 mAb were synthesized (Geneart, Regensburg, Germany) and inserted into a suitable expression vector where they were fused to the appropriate human constant heavy chain or human constant light chain. . The expression cassette in these vectors consists of the CMV promoter, intron A, along with the 5'UTR and BGH polyadenylation site. In addition, the plasmid contains the oriP region from Epstein-Barr virus for stable maintenance in HEK293 cells harboring EBV nuclear antigen (EBNA).

6.7.1.2 Transient Transfection and Production Antibodies were produced transiently in HEK293EBNA cells using a PEI-mediated transfection procedure as follows. HEK293EBNA cells are cultured in serum-free suspension in Excell culture medium containing 6mM L-glutamine. For antibody production in 500 ml shake flasks, seed 300 million HEK293EBNA cells 24 hours before transfection (thus adjust total volume for alternative scale). For transfection, cells are centrifuged at 210 x g for 10 min and the supernatant is replaced with 20 ml pre-warmed CD CHO medium. Mix the expression vector into 20 ml of CD CHO medium to give a final DNA amount of 200 μg. After adding 540 μl of PEI, the solution is mixed by vortexing for 15 seconds and then incubated for 10 minutes at room temperature. The cells are then mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours at 37°C in an incubator containing a 5% _CO2 atmosphere. After incubation, 160 ml of Ex-cell® medium (Sigma-Aldrich) containing 6mM glutamine, 1.25mM valproic acid and 12.5% Pepsoy is added and the cells are cultured for 24 hours. One day after transfection, add 12% Feed 7 (48 mL) + 3 g/L glucose. After 7 days, culture supernatants are collected for purification by centrifugation at 3000 x g for 45 min. The solution is sterile filtered (0.22 μm filter) and sodium azide is added to a final concentration of 0.01% w/v. The solution is then stored at 4°C.

6.7.1.3 Antibody Purification Secreted proteins were purified by affinity chromatography using protein A followed by size exclusion chromatography. For affinity chromatography, the supernatant was loaded onto a Protein A MabSelect SuRe column (GE Healthcare) equilibrated with 20mM sodium phosphate, 20mM sodium citrate pH 7.5. Unbound protein was removed by washing with equilibration buffer. Bound proteins were eluted using either stepwise (standard IgG) or gradient (bispecific antibody) elution made with 20mM sodium citrate, 100mM sodium chloride, 100mM glycine, pH 3.0. Ta. The pH of the collected fractions was adjusted by adding 1/10 (v/v) 0.5M sodium phosphate pH 8.0. The protein was concentrated, filtered and then loaded onto a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 20mM histidine, 140mM sodium chloride, pH 6.0.

The contents of the collection of eluted fractions were analyzed by analytical size exclusion chromatography. Therefore, 30 μl of each fraction was transferred to a TSK G3000SWXL column (Tosoh Corporation, 7.8 mm) equilibrated with 200 mM arginine, 25 mM K ₂ PO ₄ , 125 mM sodium chloride, 0.02% NaN ₃ , pH 6.7. x 30 cm). Fractions containing less than 2% oligomers were pooled and concentrated using a centrifugal concentrator (Millipore, Amicon® ULTRA-15, 30k MWCO) to a final concentration of 1-1.5 mg/ml. Purified protein was stored at -80°C.

6.7.2. Results Table 5 shows the production yield and quality of GO2 TCB antibodies.

[Example 8]
Jurkat-NFAT Reporter Assay for Monitoring Target Expression in Ex Vivo Undigested Patient-Derived Tumor Samples 6.8.1. Summary Using the Jurkat NFAT reporter assay, target expression (glycoMUC1) in ex vivo undigested primary human tumor samples was monitored using GO2 TCB.

6.8.2. Materials and Methods 6.8.2.1 Materials GO2 TCB (see Example 7)
・DP47 TCB (non-targeting negative control)
・Matrigel (item number 734-1101, Corning/VWR, Switzerland)
- Corning® Costar® Ultra-Low Attachment Multiwell Plate (Item No. CLS7007-24EA, Sigma)
- Cell culture microplate, 96 wells (item number 655098, Greiner Bio-one, Switzerland)
・RPMI1640 medium (item number 42401-018, Fisher Scientific, Schweiz)
- Jurkat medium: 2g/l D-glucose, 2g/l NaHCO ₃ , 10% FCS, 25mM HEPES, 2mM L-glutamine, 1x NEAA, 1x sodium pyruvate, 200μg/ml hygromycin B RPMI1640 medium containing Jurkat NFAT luciferase reporter cells (Promega)
- Tumor samples received from Indivumed GmbH, Germany. Samples were shipped in transport medium overnight. Approximately 24 hours after surgery, samples were cut into small pieces.

6.8.2.2 Method A 96-well cell culture microplate was prepared by adding 17 μl of cold Matrigel. Plates were incubated for 2 minutes at 37°C before tumor fragments were added (in triplicate). 33 μl of cold Matrigel was added per well and the plate was incubated again at 37° C. for 2 minutes. 100 μl (50 nM or 5 nM) of TCB antibody dilution (diluted in Jurkat medium without hygromycin but containing 2× penicillin/streptomycin) was added per well. Jurkat-NFAT reporter cells were harvested and viability assessed using ViCell. Cells were centrifuged at 350 xg for 7 minutes, after which they were resuspended in Jurkat medium without hygromycin. 50 μl of cell suspension was added per well (50,000 cells/well). The plate was incubated at 37° C. for 4 to 5 hours in a humidified incubator, after which it was removed for luminescence readout. 50 μl of ONE-Glo solution was added to each well and incubated for 10 minutes at room temperature in the dark. Luminescence was detected using a WALLAC Victor3 ELISA reader (PerkinElmer 2030) with a detection time of 5 seconds/well.

6.8.3. Results Figures 9-11 show the results from tumor samples from three patients. The results shown in Figure 9 are from tumor samples obtained from patients with malignant neoplasms of the bronchus and lungs: middle lobe, bronchial or lung, squamous cell carcinoma. The results shown in Figure 10 are from a tumor sample obtained from a patient with a malignant neoplasm of the bronchus and lungs: lower lobe, bronchus or lung, non-keratinizing squamous cell carcinoma. The results shown in Figure 11 are from a tumor sample obtained from a patient with a malignant neoplasm of the bronchus and lungs: adenocarcinoma with upper lobe, bronchial or pulmonary, acinar type. Each bar in Figures 9-11 represents the average of triplicates. Standard errors are indicated by error bars. The dotted line shows the luminescence of Jurkat NFAT cells incubated with tumor samples without any TCB. An unpaired two-tailed t-test was used for statistical analysis. P values less than 0.05 were considered significant and indicated with an asterisk ( ^* P≦0.05; ^** P≦0.001; ^*** P≦0.001). In each of Figures 9-11, tumor samples incubated with GO2 TCB exhibited significantly higher luminescence than samples incubated with DP47 negative control TCB.

[Example 9]
Characterization of GO2 TCB in vitro 6.9.1. Summary The GO2 TCB (Example 7), which recognizes tumor-specific aberrantly glycosylated MUC1, was functionally characterized in tumor cells expressing MUC1.

6.9.2. Materials and Methods 6.9.2.1 Cell Lines and PBMCs
T3M4 pfzv and MCF7 cs engineered tumor cell lines were cultured in DMEM containing 10% FCS and 2mM glutamine. MCF10A is a human non-tumorigenic breast epithelial cell line (ATCC® CRL-10317). HBEpiC are human bronchial epithelial cells (Sciencell number 3210). PBMC were isolated by gradient centrifugation using whole blood from healthy volunteers.

6.9.2.2 Target Binding by Flow Cytometry The indicated target cells were harvested using cell dissociation buffer, washed with PBS, and resuspended in FACS buffer. Antibody staining was performed in 96-well round bottom plates. 200,000 cells were seeded per well. The plate was centrifuged at 400g for 4 minutes and the supernatant was removed. Test antibodies were diluted in FACS buffer and 30 μl of antibody solution was added to cells over 30 minutes at 4°C. To remove unbound antibody, cells were washed twice with FACS buffer and then incubated with diluted secondary antibody (PE-conjugated AffiniPure F(ab')2 fragment goat anti-human IgG Fcγ fragment specific, Jackson ImmunoResearch number 109-116-170) was added. After incubation for 30 minutes at 4°C, unbound secondary antibody was removed by washing. Prior to measurements, cells were resuspended in 200 μl of FACS buffer and analyzed by flow cytometry using a BD Fortessa. Assays were performed in triplicate.

6.9.2.3 T cell-mediated tumor cell killing and T cell activation Target cells were harvested using trypsin/EDTA, counted, and checked for viability. Cells were resuspended in their respective media at a final concentration of 300,000 cells/ml. 100 μl of target cell suspension was then transferred to each well of a 96-flat bottom plate. The plates were incubated overnight at 37° C. in an incubator to allow cells to adhere to the plates. The next day, PBMC were isolated from whole blood. Blood was diluted 2:1 with PBS, layered on top of 15 ml Histopaque-1077 (no. 10771, Sigma-Aldrich) in a Leucosep tube, and centrifuged for 30 min at 450 g without brake. After centrifugation, the band containing cells was collected using a 10 ml pipette and transferred to a 50 ml tube. The tube was filled to 50 ml with PBS and centrifuged (400 g, 10 min, room temperature). The supernatant was removed and the pellet was resuspended in PBS. After centrifugation (300 g, 10 min, room temperature), the supernatant was discarded, the two tubes were pooled, and the washing step was repeated (this time centrifugation at 350 g, 10 min, room temperature). Thereafter, cells were resuspended and pellets were pooled in 50 ml PBS for cell counts. After counting the cells, they were centrifuged (350 g, 10 min, room temperature) and resuspended at 6 million cells/ml in RPMI containing 2% FCS and 2 nM glutamine. Media was removed from the plated target cells and test antibodies diluted in RPMI containing 2% FCS and 2 nM glutamine were added. 300,000 cells of effector cell solution were transferred to each well to obtain a 10:1 ratio of E:T. Target cells were lysed with Triton X-100 to determine maximum release. After 24 and 48 hours, LDH release was determined using a cytotoxicity detection kit (1644793, Roche Applied Science). Upregulation of activation markers in T cells after tumor cell killing was measured by flow cytometry. Briefly, PBMCs were collected, transferred to 96-well round bottom plates and diluted in FACS buffer for CD4APC (300514, BioLegend), CD8FITC (344704, BioLegend), CD25BV421 (302630, BioLegend), CD69PE (3 10906 , BioLegend) antibody. After incubation for 30 minutes at 4°C, cells were washed twice with FACS buffer. Cells were resuspended in 200 μl of FACS buffer before measuring fluorescence using a BD Fortessa II. Assays were performed in triplicate.

6.9.2.4 Cytokine/chemokine release by cytometry bead array Cytokine/chemokine secretion in the supernatant was measured by flow cytometry using a cytometry bead array (CBA) according to the manufacturer's guidelines. Supernatants from T cell-mediated lethal assays were collected and stored at -20°C. The supernatant was then thawed and tested according to the manufacturer's instructions. The following CBA kits (BD Biosciences) were used: CBA Human Interferon Gamma (IFNγ) Flex Set (E7), CBA Human Granzyme B Flex Set (D7), CBA Human IL6 Flex Set (A7), CBA Human IL8 Flex Set ( A9), CBA human IL10 Flex set (B7) and CBA human tumor necrosis factor (TNF) Flex set (D9). Samples were measured using a BD FACS Canto II and analysis was performed using Diva software (BD Biosciences). Assays were performed in triplicate.

6.9.3. Results We confirmed binding of GO2 TCB to breast cancer cell line MCF7 and pancreatic cancer cell line T3M4, both engineered to express aberrantly glycosylated MUC1 (Figure 12). Freshly isolated PBMCs were then used to test the activity of GO2 TCB in both tumor cell lines, MCF7 and T3M4 (Figures 13 and 14). Tumor cell killing of both cell lines was detected after 24 hours and was even stronger after 48 hours. Concomitantly, a strong activation of CD4 and CD8 T cells occurred as determined by the upregulation of the two activation markers CD25 and CD69 and the release of IL6, IL8, IL10, IFNγ, TNFα and granzyme B into the supernatant. Ta. Each non-targeted TCB is included as a negative control.

To demonstrate that GO2 TCB does not bind to normally glycosylated MUC1 on epithelial cells, we demonstrated binding to MCF10A, a human non-tumorigenic breast epithelial cell line, and HBEpiC, a primary human bronchial epithelial cell line. was tested. As a positive control, HMFG1 TCB was included, which does not discriminate between MUC1 expressed on normal cells and MUC1 expressed on tumor cells. HFMG1 TCB was found to bind to both tested cells, confirming the expression of MUC1, whereas GO2 TCB was unable to bind to these cells (Figure 15). In addition, GO2 TCB was tested to see if it induced lethality or T cell activation in the presence of normal epithelial cells expressing MUC1. When this was tested in MCF10A cells, there was no detectable lethality or T cell activation with GO2 TCB, whereas HMFG1 TCB induced T cell activation in addition to lethality (Figure 16).

[Example 10]
Functional characterization of GO2 and GO2 TCB antibodies by surface plasmon resonance 6.10.1. Summary GO2 and GO2 TCB (Example 7) were characterized by surface plasmon resonance.

6.10.2. Materials and Methods 6.10.2.1 Binding of GO2 and GO2 TCB to Immobilized Glycopeptides Binding of GO2 antibodies and GO2 TCB to human and cynomolgus monkey glycopeptides (Table 6) was determined by surface plasmon resonance (SPR). Evaluated by. All SPR experiments were performed on a Biacore T200 at 25 °C with HBS-EP (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20 as running buffer). , Biacore, Freiburg/Germany).

Biotinylated glycopeptides were dissolved in PBS to a final concentration between 0.9 and 1.8 mg/ml (Table 6). Biotinylated glycopeptides were coupled directly to the flow cell of a streptavidin (SA) sensor chip. A fixed level of up to 880 resonance units (RU) was used. GO2 antibody or GO2 TCB was injected at a concentration of 1000 nM over 240 seconds using a flow of 30 μL/min through the flow cell (Figure 17). Dissociation was monitored for 500 seconds. Bulk refractive index differences were corrected by subtracting the response obtained in a reference flow cell without immobilized proteins.

6.10.2.2 Binding activity of GO2 and GO2 TCB to immobilized glycopeptide The binding activity of GO2 and GO2 TCB was evaluated by surface plasmon resonance (SPR). All SPR experiments were performed on a Biacore T200 at 25 °C with HBS-EP (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20 as running buffer). , Biacore, Freiburg/Germany). Biotinylated glycopeptides (Table 6) were coupled directly to the flow cell of a streptavidin (SA) sensor chip. A fixed level of up to 200 resonance units (RU) was used.

GO2 antibody or GO2 TCB was injected at concentrations ranging from 3.9 to 1000 nM (1:2 dilution) over 120 seconds using a flow of 30 μL/min through the flow cell. Dissociation was monitored for 400 seconds. Bulk refractive index differences were corrected by subtracting the response obtained in a reference flow cell without immobilized proteins. Despite the bivalent nature of the interaction, the KD was obtained by fitting the curve to 1:1 Langmuir binding using Biaeval software (GE Healthcare). The "apparent" KD can therefore only be used for comparison purposes.

6.10.3. Results As can be seen from the sensorgram in Figure 18, GO2 antibody (Figure 18A) and GO2 TCB (Figure 18B) bind both human and cynomolgus monkey glycopeptides. GO2 antibody and GO2 TCB bind to cynomolgus monkey glycopeptides with higher avidity than human glycopeptides.

As can be seen from Figure 19, the binding of divalent GO2 binders (IgG, TCB) to human glycopeptides is three orders of magnitude nanomolar (Figures 19A and 19C), whereas to cynomolgus monkey glycopeptides. The binding is two orders of magnitude nanomolar (FIGS. 19B and 19D).

[Example 11]
Exploratory single-dose pharmacokinetic and tolerability study of GO2 TCB in cynomolgus monkeys 6.11.1. Summary The purpose of this study was to determine the pharmacokinetics and tolerability of the GO2 TCB described in Example 7 when given by a single intravenous injection to cynomolgus monkeys.

6.11.2. Materials and Methods 6.11.2.1 Preparation of GO2 TCB Frozen GO2 TCB (2.12 mg/mL) and formulation buffer (20 mM histidine, 140 mM NaCl, 0.01% Tween 20; pH 6.0) Thaw the stock solution overnight in a refrigerator set to be maintained at 4°C. Test item dosing formulations are prepared under sterile conditions by dilution with formulation buffer to the appropriate concentration to meet dose level requirements.

Dosage formulations are prepared within 2 hours prior to injection and stored at room temperature until use. Polypropylene containers are used to prepare and store the dosage formulation to prevent adsorption. The dosage formulation should not be filtered, nor should it be agitated or shaken. Any mixing is done by either gentle pipetting or gentle shaking of the container.

6.11.2.2 Animals Cynomolgus monkeys (Macaca fascicularis), 2-4 years old and weighing less than 4 kg, are used for the study. Animals are acclimated to the testing facility, the Primate Toxicology Facility, for at least 6 weeks prior to the start of dosing.

During the week prior to the start of dosing, animals were tested for participation in the experiment based on a thorough veterinary examination (performed immediately upon arrival), clinical observations, body weight profile and clinicopathological investigation. Get approved.

Animals selected for study are randomly assigned to cages based on group suitability information provided and then individual study numbers provided. Animals are assigned to cages in groups of up to five.

6.11.2.3 Management Socially isolate animals, if possible, in groups of up to five by sex in two-story group enclosures, with a 1.61 x 1.66 x 2.5 m, and the upper floor measures 1.61 x 1.66 x 2.03 m. The base material is wood shavings. There are no known contaminants in the substrate that would be expected to interfere with research objectives.

Targeting conditions of the animal room environment are as follows:
Temperature: 18-24℃
Humidity: 40-70%
Ventilation: Minimum of 10 air exchanges per hour Light cycle: 12 hours light and 12 hours dark (except when interrupted by research procedures/activities).

Automatic control of temperature and humidity is provided, which is continuously monitored and recorded. Automatic control of the light cycle is performed.

Special Diets Service (SDS) MP(E) Short SQC Diet will be provided as a daily ration throughout the study. A food ration of approximately 200 grams per animal is provided once a day. There are no known contaminants in the feed that would be expected to interfere with research objectives. Animals receive unlimited water from communal sources. There are no known contaminants in the water that would be expected to interfere with research objectives.

Enrich your animal's indoor environment so that social interaction, play, and exploration are encouraged. The animals have a safe place and materials such as plastic toys, balls, climbing frames and stainless steel mirrors. These are replaced frequently to reduce habituation. Before replacement, all toys and climbing frames are thoroughly cleaned to avoid cross-contamination. Animals are also provided with a variety of other rewards, such as feed mixes, vegetables, nuts, biscuits, and fruit, usually on a daily basis.

Throughout the course of the study, animals will be examined by veterinary staff if available for veterinary care and as warranted by clinical signs or other changes.

6.11.2.4 Experimental Design One male and one female animal will be administered GO2 TCB.

GO2 TCB is administered to suitable animals by a single slow intravenous bolus injection (1-2 minutes) in the saphenous or tail vein at least 8 days apart. This is staggered so that only one male and one female receives the new dose level on any given day. Increase or decrease the dose in the next dose group based on observations from previous dose levels (including clinicopathological data). Naive animals are used for each dose level. Doses are given using a syringe fitted with a Vygon needle.

Animals are necropsied approximately 72 hours after dosing (after the last scheduled sample is taken). For all animals that have to be terminated before the scheduled date due to severe clinical symptoms, a complete panel of clinicopathology (additional sampling before termination if possible) and histopathology will be analyzed. Ru.

The intravenous route of administration was chosen for this study as it is a route of possible clinical application. Low and high dose levels were chosen to cover a clinically relevant dose range and to minimize possible injury to the animals. The low dose was chosen based on experience in cynomolgus monkeys with similar T cell bispecific antibodies of similar potency, and the high dose corresponds to a 3-fold increase.

6.11.3. Results GO2 TCB is tolerated at the doses tested.

7. Specific Embodiments, Reference Citations Although various specific embodiments have been illustrated and described, it is to be understood that various changes may be made without departing from the spirit and scope of the disclosure. Ru. The present disclosure is illustrated by the numbered embodiments described below.

1. a. preferentially binds to the glycoMUC1 epitope that is overexpressed on cancer cells compared to normal cells;
b. An anti-glycoMUC1 antibody or antigen that competes with an antibody or antigen-binding fragment comprising a heavy chain variable (VH) sequence of SEQ ID NO: 3 and a light chain variable (VL) sequence of SEQ ID NO: 4 for binding to breast cancer cell lines MCF7 or T47D. Combined fragment.

2. a. The MUC1 tandem repeat (VTSAPDTRPAPGSTAPPAHG) ₃ (hereinafter referred to as "first epitope") was glycosylated in vitro using purified recombinant human glycosyltransferases GalNAc-T1, GalNAc-T2, and GalNAc-T4. ); and
b. An anti-glycoMUC1 antibody or antigen that competes with an antibody or antigen-binding fragment comprising a heavy chain variable (VH) sequence of SEQ ID NO: 3 and a light chain variable (VL) sequence of SEQ ID NO: 4 for binding to breast cancer cell lines MCF7 or T47D. Combined fragment.

3. Complementarity determining region (CDR) H1 containing the amino acid sequence of SEQ ID NO: 33, CDR-H2 containing the amino acid sequence of SEQ ID NO: 29, CDR-H3 containing the amino acid sequence of SEQ ID NO: 25, CDR containing the amino acid sequence of SEQ ID NO: 8 -L1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 31.

4. The anti-glycoMUC1 antibody or antigen-binding fragment of embodiment 3, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 5.

5. The anti-glycoMUC1 antibody or antigen-binding fragment of embodiment 3, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 23.

6. The anti-glycoMUC1 antibody or antigen-binding fragment of embodiment 3, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 28.

7. The anti-glycoMUC1 antibody or antigen-binding fragment of embodiment 3, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 32.

8. The anti-glycoMUC1 antibody or antigen-binding fragment according to any one of embodiments 3 to 7, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6.

9. The anti-glycoMUC1 antibody or antigen-binding fragment according to any one of embodiments 3 to 7, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO: 24.

10. The anti-glycoMUCl antibody or antigen-binding fragment according to any one of embodiments 3 to 9, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7.

11. The anti-glycoMUC1 antibody or antigen-binding fragment according to any one of embodiments 3 to 10, wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO: 30.

12. The anti-glycoMUC1 antibody or antigen-binding fragment according to any one of embodiments 3 to 10, wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO: 26.

13. The anti-glycoMUC1 antibody or antigen-binding fragment according to any one of embodiments 3 to 12, wherein CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27.

14. The anti-glycoMUC1 antibody or antigen-binding fragment according to any one of embodiments 3 to 13, wherein CDR-L3 comprises the amino acid sequence of SEQ ID NO: 10.

15. The anti-glycoMUC1 antibody or antigen-binding fragment according to Embodiment 1 or 2, wherein the VH comprises the complementarity determining regions (CDRs) of SEQ ID NOs: 5 to 7, and the VL comprises the CDRs of SEQ ID NOs: 8 to 10. .

16. The anti-glycoMUC1 antibody according to embodiment 1 or embodiment 2, wherein the VH comprises the complementarity determining regions (CDRs) of SEQ ID NOs: 23-25, and the VL comprises the CDRs of SEQ ID NOs: 26, 27, and 10, or Antigen-binding fragment.

17. The anti-glycosylation agent of embodiment 1 or embodiment 2, wherein the VH comprises the complementarity determining regions (CDRs) of SEQ ID NOs: 28, 29, and 25, and the VL comprises the CDRs of SEQ ID NOs: 30, 9, and 31. MUC1 antibody or antigen binding fragment.

18. The anti-glycoMUC1 antibody or antigen-binding fragment according to any one of embodiments 1 to 17, which is a chimeric or humanized antibody.

19. Any of embodiments 1-18, wherein the VH comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 3 and the VL comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 4. 1. The anti-glycoMUC1 antibody or antigen-binding fragment according to item 1.

20. Any of embodiments 1-18, wherein the VH comprises an amino acid sequence that has at least 97% sequence identity with SEQ ID NO: 3 and the VL comprises an amino acid sequence that has at least 97% sequence identity with SEQ ID NO: 4. 1. The anti-glycoMUC1 antibody or antigen-binding fragment according to item 1.

21. Any of embodiments 1 to 18, wherein the VH comprises an amino acid sequence that has at least 99% sequence identity with SEQ ID NO: 3 and the VL comprises an amino acid sequence that has at least 99% sequence identity with SEQ ID NO: 4. 1. The anti-glycoMUC1 antibody or antigen-binding fragment according to item 1.

22. The anti-glycoMUC1 antibody or antigen-binding fragment according to embodiment 1 or embodiment 2, wherein the VH comprises the amino acid sequence of SEQ ID NO: 3 and the VL comprises the amino acid sequence of SEQ ID NO: 4.

23. 23. The anti-glycoMUC1 antibody or antigen-binding fragment of any of embodiments 1-22, which is multivalent.

24. 23. The anti-glycoMUC1 antibody or antigen binding fragment according to any of embodiments 1 to 22, which is in the form of a single chain variable fragment (scFv).

25. The anti-glycoMUC1 antibody or antigen-binding fragment of embodiment 24, wherein the scFv comprises a heavy chain variable fragment N-terminal to a light chain variable fragment.

26. The anti-glycoMUC1 antibody or antigen-binding fragment of embodiment 24, wherein the scFv heavy chain variable fragment and light chain variable fragment are covalently linked to a linker sequence of 4 to 15 amino acids.

27. 23. The anti-glycoMUC1 antibody or antigen-binding fragment according to any of embodiments 1 to 22, which is in the form of a multispecific antibody.

28. 28. The anti-glycoMUC1 antibody or antigen-binding fragment of embodiment 27, wherein the multispecific antibody is a bispecific antibody that binds a second epitope that is different from the first epitope.

29. The anti-glycoMUC1 antibody of embodiment 28, wherein the bispecific antibody is a CrossMab, a Fab arm-swapped antibody, a bispecific T cell derivative (BiTE), or a dual affinity retargeting molecule (DART). or antigen-binding fragments.

30. The anti-glycoMUC1 antibody or antigen-binding fragment of embodiment 29, wherein the bispecific antibody is a CrossMab.

31. 31. The anti-glycoMUC1 antibody or antigen-binding fragment of embodiment 30, wherein the bispecific antibody is CrossMab ^FAB .

32. The anti-glycoMUCl antibody or antigen-binding fragment of embodiment 30, wherein the bispecific antibody is CrossMab ^VH-VL .

33. The anti-glycoMUC1 antibody or antigen-binding fragment of embodiment 30, wherein the bispecific antibody is CrossMab ^CH1-CL .

34. The anti-glycoMUC1 antibody or antigen-binding fragment of embodiment 29, wherein the bispecific antibody is a Fab arm-swapped antibody.

35. The anti-glycoMUC1 antibody or antigen-binding fragment of embodiment 29, wherein the bispecific antibody is a dual affinity retargeting molecule (DART).

36. The anti-glycoMUC1 antibody or antigen-binding fragment of embodiment 29, wherein the bispecific antibody is a bispecific T cell derivative (BiTE).

37. 36. The anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 28-35, wherein the second epitope is a MUC1 epitope.

38. 36. The anti-glyco-MUC1 antibody or antigen-binding fragment of any one of embodiments 28-35, wherein the second epitope is a MUC1 epitope that is overexpressed on cancer cells compared to normal cells.

39. 37. The anti-glycoMUC1 antibody or antigen-binding fragment of any one of embodiments 28-36, wherein the second epitope is a T cell epitope.

40. The anti-glycoMUC1 antibody or antigen-binding fragment of embodiment 39, wherein the T cell epitope comprises a CD3 epitope, a CD8 epitope, a CD16 epitope, a CD25 epitope, a CD28 epitope, or an NKG2D epitope.

41. 41. The anti-glycoMUCl antibody or antigen-binding fragment of embodiment 40, wherein the T cell epitope comprises a CD3 epitope, optionally an epitope present on human CD3.

42. The anti-glycoMUCl antibody or antigen-binding fragment of embodiment 41, wherein the CD3 epitope comprises a CD3 gamma epitope, a CD3 delta epitope, a CD3 epsilon epitope, or a CD3 zeta epitope.

43. 43. The anti-glycoMUC1 antibody or antigen-binding fragment of any one of embodiments 1-42, conjugated to a detectable moiety.

44. 44. The anti-glycoMUC1 antibody or antigen-binding fragment of embodiment 43, wherein the detectable marker is an enzyme, a radioisotope, or a fluorescent label.

45. A bispecific antibody comprising a first antigen binding domain that binds CD3 (optionally human CD3) and a second antigen binding domain that binds glycoMUC1, the bispecific antibody comprising a breast cancer cell line. competes for binding to MCF7 or T47D with an antibody or antigen-binding fragment comprising a heavy chain variable (VH) sequence of SEQ ID NO: 3 and a light chain variable (VL) sequence of SEQ ID NO: 4, the first antigen-binding domain comprising: A heavy chain variable region comprising heavy chain CDR-H1 of SEQ ID NO: 34, CDR-H2 of SEQ ID NO: 35, and CDR-H3 of SEQ ID NO: 36; and light chain CDR-L1 of SEQ ID NO: 37, CDR- of SEQ ID NO: 38. A bispecific antibody comprising a light chain variable region comprising L2 and CDR-L3 of SEQ ID NO: 39.

46. The second antigen-binding domain comprises (i) a heavy chain variable region comprising CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6, and CDR-H3 of SEQ ID NO: 7; and a light chain CDR of SEQ ID NO: 8; -L1, a light chain variable region comprising CDR-L2 of SEQ ID NO: 9 and CDR-L3 of SEQ ID NO: 10.

47. The first antigen binding domain has a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 40, and the amino acid sequence of SEQ ID NO: 41. 47. The bispecific antibody of embodiment 45 or embodiment 46, comprising light chain variable region sequences that are at least about 95%, 96%, 97%, 98%, 99% or 100% identical.

48. 48. The bispecific antibody of embodiment 47, wherein the first antigen binding domain comprises the heavy chain variable region sequence of SEQ ID NO: 40 and the light chain variable region sequence of SEQ ID NO: 41.

49. The second antigen binding domain has a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3, and the amino acid sequence of SEQ ID NO: 4. 49. The bispecific antibody of any one of embodiments 45-48, comprising light chain variable region sequences that are at least about 95%, 96%, 97%, 98%, 99% or 100% identical.

50. 50. The bispecific antibody of embodiment 49, wherein the second antigen binding domain comprises the heavy chain variable region sequence of SEQ ID NO: 3 and the light chain variable region sequence of SEQ ID NO: 4.

51. Bispecific antibody according to any one of embodiments 45 to 50, wherein the first and/or second antigen binding domain is a Fab molecule.

52. 52. The bispecific antibody of embodiment 51, wherein the first antigen binding domain is a crossed Fab molecule in which either the variable or constant regions of the Fab light chain and Fab heavy chain are exchanged.

53. The first and second antigen-binding domains of the bispecific antibody are both Fab molecules, and in one of the antigen-binding domains (particularly the first antigen-binding domain), the variable domain VL of the Fab light chain and the Fab heavy chain and VH are replaced with each other,
a. In the constant domain CL of the first antigen-binding domain, the amino acid at position 124 is replaced with a positively charged amino acid (numbering according to Kabat), and in the constant domain CH1 of the first antigen-binding domain, the amino acid at position 147 is replaced with a positively charged amino acid (numbering according to Kabat). the amino acid or the amino acid at position 213 is replaced with a negatively charged amino acid (numbering according to the Kabat EU index); or b. In the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is replaced with a positively charged amino acid (numbering according to Kabat), and in the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 is replaced with a positively charged amino acid (numbering according to Kabat). The amino acid or amino acid at position 213 is replaced with a negatively charged amino acid (numbering according to the Kabat EU index);
53. The bispecific antibody according to embodiment 52, wherein the constant domains CL and CH1 of the antigen binding domain with VH/VL exchange are not replaced with each other.

54. a. In the constant domain CL of the first antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat); In the constant domain CH1 of the antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E), or aspartic acid (D) (numbering according to the Kabat EU index); or b ．． In the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat); Embodiments in which in the constant domain CH1 of the antigen binding domain, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index) 53. The bispecific antibody according to 53.

55. In the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat); Embodiments in which in the constant domain CH1 of the antigen binding domain, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index) 54. The bispecific antibody according to 54.

56. In the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat); 56, wherein in the constant domain CH1 of the antigen binding domain, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index). Heavy specific antibody.

57. In the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat); The amino acids are independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 is The amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index); 56. The bispecific antibody according to embodiment 55, wherein

58. In the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat), and the amino acid at position 123 is substituted with lysine (K) ( In the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to Kabat EU index), and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to Kabat EU index). E) (numbering according to Kabat EU index).

59. In the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat), and the amino acid at position 123 is substituted with arginine (R) (numbering according to Kabat). In the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to Kabat EU index), and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to Kabat EU index). E) (numbering according to Kabat EU index).

60. 60. The bispecific antibody according to any one of embodiments 53 to 59, wherein the constant domain CL of the second antigen binding domain is a constant domain CL of the kappa isotype.

61. 61. The bispecific antibody according to any one of embodiments 45 to 60, wherein the first and second antigen binding domains are fused to each other, optionally via a peptide linker.

62. the first and second antigen-binding domains are each Fab molecules, (i) the second antigen-binding domain is at the C-terminus of the Fab heavy chain and the N-terminus of the Fab heavy chain of the first antigen-binding domain; or (ii) the first antigen-binding domain is fused to the C-terminus of the Fab heavy chain and the second antigen-binding domain is fused to the N-terminus of the Fab heavy chain. Bispecific antibody according to Form 61.

63. 63. The bispecific antibody according to any one of embodiments 45-62, wherein the bispecific antibody provides monovalent binding to CD3.

64. 64. The bispecific antibody of embodiment 63, comprising two antigen binding domains that specifically bind to glycoMUC1.

65. 65. The bispecific antibody of embodiment 64, wherein the two antigen binding domains that specifically bind to glycoMUC1 contain the same amino acid sequence.

66. 66. The bispecific antibody of any one of embodiments 45-65, wherein the bispecific antibody further comprises an Fc domain composed of a first and a second subunit.

67. 67. The bispecific antibody of embodiment 66, wherein the Fc domain is an IgG Fc domain.

68. 68. The bispecific antibody of embodiment 67, wherein the Fc domain is an _IgGi Fc domain.

69. 68. The bispecific antibody of embodiment 67, wherein the Fc domain is an _IgG4 Fc domain.

70. Bispecific antibody according to embodiment 69, wherein the IgG ₄ Fc domain comprises an amino acid substitution at position S228 (Kabat EU Index numbering), preferably the amino acid substitution S228P.

71. 67. The bispecific antibody of embodiment 66, wherein the Fc domain is a human Fc domain.

72. 72. The bispecific antibody of embodiment 71, wherein the Fc domain is a human IgGi _Fc domain, and the human IgGi Fc domain optionally comprises SEQ ID NO: ₄₂ .

73. the first, second, and, if present, third antigen-binding domain are each a Fab molecule, (a) (i) the second antigen-binding domain is at the C-terminus of the Fab heavy chain; an antigen-binding domain fused to the N-terminus of the Fab heavy chain, the first antigen-binding domain being fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, or (ii) the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain; (b) the third antigen-binding domain, if present, is either fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain; 73. The bispecific antibody of any one of embodiments 66-72, fused to the N-terminus of the second subunit of the domain.

74. 74. The bispecific antibody of any one of embodiments 66-73, wherein the Fc domain comprises a modification that promotes association of the first and second subunits of the Fc domain.

75. 75. The bispecific antibody according to any one of embodiments 66-74, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and/or effector functions.

76. a. a first antigen-binding domain that specifically binds to CD3, the first antigen-binding domain being a crossed Fab molecule, comprising either the variable or constant region of a Fab light chain and a Fab heavy chain, preferably a variable a first antigen-binding domain in which the regions have been exchanged;
b. second and third antigen-binding domains that specifically bind to glycoMUC1, comprising heavy chain CDR-H1 of SEQ ID NO: 5, CDR-H2 of SEQ ID NO: 6, and CDR-H3 of SEQ ID NO: 7; and a light chain variable region comprising light chain CDR-L1 of SEQ ID NO: 8, CDR-L2 of SEQ ID NO: 9 and CDR-L3 of SEQ ID NO: 10, wherein the second and third antigen binding domains are second and third antigen binding domains, each being a Fab molecule;
c. comprising an Fc domain composed of a first and second subunit capable of stable association;
the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain; , fused to the N-terminus of the first subunit of the Fc domain, and the third antigen-binding domain is fused to the N-terminus of the second subunit of the Fc domain, at the C-terminus of the Fab heavy chain. , the bispecific antibody according to embodiment 45.

77. The first antigen-binding domain comprises a heavy chain CDR-H1 of SEQ ID NO: 34, a CDR-H2 of SEQ ID NO: 35, and a CDR-H3 of SEQ ID NO: 36; and a light chain CDR-H3 of SEQ ID NO: 37; 78. The bispecific antibody of embodiment 77, comprising a light chain variable region comprising L1, CDR-L2 of SEQ ID NO: 38, and CDR-L3 of SEQ ID NO: 39.

78. The first antigen binding domain has a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 40, and the amino acid sequence of SEQ ID NO: 41. 78. The bispecific antibody of embodiment 77, comprising light chain variable region sequences that are at least about 95%, 96%, 97%, 98%, 99% or 100% identical.

79. 79. The bispecific antibody of embodiment 78, wherein the first antigen binding domain comprises the heavy chain variable region sequence of SEQ ID NO: 40 and the light chain variable region sequence of SEQ ID NO: 41.

80. The second and third antigen binding domains have heavy chain variable region sequences that are at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3, and the amino acid sequence of SEQ ID NO: 4. The bispecific antibody of any one of embodiments 76-79, comprising a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence. .

81. 81. The bispecific antibody of embodiment 80, wherein the second and third antigen binding domains comprise the heavy chain variable region of SEQ ID NO: 3 and the light chain variable region of SEQ ID NO: 4.

82. 82. The bispecificity of any one of embodiments 76-81, wherein the Fc domain comprises any of the features described in Sections 5.1 and 5.2 with respect to Fc domains, alone or in combination. antibody.

83. 83. The bispecific antibody according to any one of embodiments 76-82, wherein the antigen binding domain and the Fc region are fused to each other by a peptide linker.

84. 84. The bispecific antibody according to embodiment 83, wherein the peptide linker comprises a peptide linker as in SEQ ID NO: 45 and/or SEQ ID NO: 46.

85. In the constant domain CL of the second and third Fab molecules, the amino acid at position 124 is replaced with lysine (K) (numbering according to Kabat), and the amino acid at position 123 is replaced with lysine (K) or arginine (R ), preferably substituted with arginine (R) (numbering according to Kabat), and in the constant domain CH1 of the second and third Fab molecules of (ii), the amino acid at position 147 is substituted with glutamic acid (E). (numbering according to Kabat EU index), the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to Kabat EU index) according to any one of embodiments 76 to 84. Bispecific antibodies.

86. A polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 43, at least 80%, 85 to the sequence of SEQ ID NO: 44. %, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 45, at least 80%, 85%, 90%, 95%, 96%; A polypeptide comprising a sequence that is 97%, 98%, or 99% identical, and at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 46. 86. The bispecific antibody of any one of embodiments 76-85, comprising a polypeptide comprising a sequence.

87. The bispecific antibody comprises a polypeptide comprising the sequence SEQ ID NO: 43, a polypeptide comprising the sequence SEQ ID NO: 44, a polypeptide comprising the sequence SEQ ID NO: 45, and a polypeptide comprising the sequence SEQ ID NO: 46, Bispecific antibody according to embodiment 86.

88. 88. The bispecific antibody of embodiment 87, comprising two polypeptides comprising the sequence of SEQ ID NO: 43.

89. 89. The bispecific antibody according to any one of embodiments 45-88, conjugated to a detectable moiety.

90. 90. The bispecific antibody of embodiment 89, wherein the detectable marker is an enzyme, a radioisotope, or a fluorescent label.

91. an anti-glycoMUC1 antibody or antigen-binding fragment according to any one of embodiments 1 to 44, or a bispecific according to any one of embodiments 45 to 90, operably linked to at least a second amino acid sequence; A fusion protein containing the amino acid sequence of a sex antibody.

92. 92. The fusion protein of embodiment 91, wherein the second amino acid sequence is that of 4-1BB, CD3-zeta, or a fragment thereof.

93. 92. The fusion protein of embodiment 91, wherein the second amino acid sequence is the amino acid sequence of the fusion peptide.

94. The fusion protein of embodiment 93, wherein the fusion peptide is a CD28-CD3-zeta or 4-1BB(CD137)-CD3-zeta fusion peptide.

95. 92. The fusion protein of embodiment 91, wherein the second amino acid sequence is an amino acid sequence of a modulator of T cell activation or a fragment thereof.

96. 96. The fusion protein of embodiment 95, wherein the modulator of T cell activation is IL-15 or IL-15Ra.

97. A chimeric antigen receptor (CAR) comprising an scFv according to any one of embodiments 24-26.

98. 98. The CAR of embodiment 97, comprising, in order from amino terminus to carboxy terminus, a human CD8 leader peptide, a scFv, a human CD8 hinge domain, a human CD8 transmembrane domain, and a CD3-zeta signaling domain.

99. an anti-glycoMUC1 antibody or antigen-binding fragment according to any one of embodiments 1 to 44, or a bispecific antibody according to any one of embodiments 45 to 90, conjugated to a cytotoxic agent; or An antibody-drug conjugate comprising a fusion protein according to any one of embodiments 91-96.

100. The antibody of embodiment 99, wherein the cytotoxic agent is an auristatin, a DNA minor groove binder, an alkylating agent, an enediyne, a lexitropsin, a duocarmycin, a taxane, a dostatin, a maytansinoid, or a vinca alkaloid. -Drug conjugates.

101. 101. The antibody-drug conjugate of embodiment 100, wherein the anti-glycoMUC1 antibody or antigen-binding fragment or bispecific antibody is conjugated to a cytotoxic agent via a linker.

102. The antibody-drug conjugate of embodiment 101, wherein the linker is cleavable under intracellular conditions.

103. 103. The antibody-drug conjugate of embodiment 102, wherein the cleavable linker is cleavable by an intracellular protease.

104. 104. The antibody-drug conjugate of embodiment 103, wherein the linker comprises a dipeptide.

105. The antibody-drug conjugate according to embodiment 104, wherein the dipeptide is val-cit or phe-lys.

106. 103. The antibody-drug conjugate of embodiment 102, wherein the cleavable linker is hydrolyzable at a pH of less than 5.5.

107. 107. The antibody-drug conjugate of embodiment 106, wherein the hydrolyzable linker is a hydrazone linker.

108. 103. The antibody-drug conjugate of embodiment 102, wherein the cleavable linker is a disulfide linker.

109. an anti-glycoMUC1 antibody or antigen-binding fragment according to any one of embodiments 1 to 44, or a bispecific antibody according to any one of embodiments 45 to 90, any one of embodiments 91 to 96 or a nucleic acid comprising the coding region of a CAR according to embodiment 97 or embodiment 98.

110. 110. The nucleic acid of embodiment 109, wherein the coding region is codon-optimized for expression in human cells.

111. A vector comprising a nucleic acid according to embodiment 109 or embodiment 110.

112. The vector of embodiment 111, which is a viral vector.

113. 113. The vector of embodiment 112, wherein the viral vector is a lentiviral vector.

114. A host cell engineered to express a nucleic acid according to embodiment 109 or embodiment 110.

115. The host cell of embodiment 114 is a human T cell engineered to express a CAR of embodiment 97 or embodiment 98.

116. A host cell comprising a vector according to any one of embodiments 111-113.

117. 117. The host cell of embodiment 116, wherein the host cell is a T cell and the vector encodes a CAR of embodiment 97 or embodiment 98.

118. (a) the anti-glycoMUC1 antibody or antigen-binding fragment of any one of embodiments 1-44, the bispecific antibody of any one of embodiments 45-90, any of embodiments 91-96; a fusion protein according to one of embodiments 97 or 98, an antibody-drug conjugate according to any one of embodiments 99 to 108, a nucleic acid according to embodiment 109 or embodiment 110. , a vector according to any one of embodiments 111 to 113, or a host cell of an embodiment according to any one of embodiments 114 to 117, and (b) a physiologically suitable buffer, adjuvant, or A pharmaceutical composition comprising a diluent.

119. A method of treating cancer, comprising administering to a subject in need thereof an effective amount of the anti-glycoMUC1 antibody or antigen-binding fragment of any of embodiments 1-44, of any of embodiments 45-90. a fusion protein according to any one of embodiments 91-96, a CAR according to embodiment 97 or 98, a bispecific antibody according to any one of embodiments 99-108. a nucleic acid according to embodiment 109 or embodiment 110, a vector according to any one of embodiments 111 to 113, an antibody-drug conjugate according to any one of embodiments 114 to 117. A method comprising administering a host cell or a pharmaceutical composition according to embodiment 118.

120. 120. The method of embodiment 119, wherein the subject has breast cancer, non-small cell lung cancer, prostate cancer, pancreatic cancer, esophageal cancer, or colorectal cancer.

121. 45. A method of detecting cancer in a biological sample, the method comprising: contacting the sample with an anti-glycoMUC1 antibody or antigen-binding fragment according to any one of embodiments 1-44; A method comprising detecting binding of an antigen binding fragment.

122. 122. The method of embodiment 121, further comprising quantifying binding of the anti-glycoMUC1 antibody or antigen binding fragment.

123. 123. A method according to embodiment 121 or embodiment 122, wherein the binding is compared to a normal tissue control as a negative/baseline control and/or compared to a cancerous tissue control as a positive control.

All publications, patents, patent applications, and other documents cited in this application are each individually indicated to be incorporated by reference for all purposes. are incorporated herein by reference in their entirety for all purposes. In the event that there is a discrepancy between the teachings of one or more of the references incorporated herein and the disclosure of the present invention, the teachings of the present specification are intended.

The present invention includes the following embodiments.
[1]
a. preferentially binds to the glycoMUC1 epitope that is overexpressed on cancer cells compared to normal cells;
b. An anti-glycoMUC1 antibody or antigen that competes with an antibody or antigen-binding fragment comprising a heavy chain variable (VH) sequence of SEQ ID NO: 3 and a light chain variable (VL) sequence of SEQ ID NO: 4 for binding to breast cancer cell lines MCF7 or T47D. Combined fragment.
[2]
a. The MUC1 tandem repeat (VTSAPDTRPAPGSTAPPAHG) ₃ (hereinafter referred to as "first epitope") was glycosylated in vitro using purified recombinant human glycosyltransferases GalNAc-T1, GalNAc-T2, and GalNAc-T4. ); and
b. competes with an antibody or antigen-binding fragment comprising a heavy chain variable (VH) sequence of SEQ ID NO: 3 and a light chain variable (VL) sequence of SEQ ID NO: 4 for binding to breast cancer cell lines MCF7 or T47D;
Anti-glycoMUC1 antibody or antigen binding fragment.
[3]
Complementarity determining region (CDR) H1 containing the amino acid sequence of SEQ ID NO: 33, CDR-H2 containing the amino acid sequence of SEQ ID NO: 29, CDR-H3 containing the amino acid sequence of SEQ ID NO: 25, CDR containing the amino acid sequence of SEQ ID NO: 8 -L1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 31, the anti-glycoMUC1 antibody or antigen-binding fragment according to [1] or [2] above.
[4]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [3] above, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 5.
[5]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [3] above, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 23.
[6]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [3] above, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 28.
[7]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [3] above, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 32.
[8]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [3] above, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6.
[9]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [3] above, wherein CDR-H2 comprises the amino acid sequence of SEQ ID NO: 24.
[10]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [3] above, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7.
[11]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [3] above, wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO: 30.
[12]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [3] above, wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO: 26.
[13]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [3] above, wherein CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27.
[14]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [3] above, wherein CDR-L3 comprises the amino acid sequence of SEQ ID NO: 10.
[15]
The anti-glycoMUC1 antibody or the anti-glycoMUC1 antibody according to [1] or [2] above, wherein the VH includes the complementarity determining regions (CDRs) of SEQ ID NOs: 5 to 7, and the VL includes CDRs of SEQ ID NOs: 8 to 10. Antigen-binding fragment.
[16]
The anti-glycosylation agent according to [1] or [2] above, wherein the VH includes the complementarity determining regions (CDRs) of SEQ ID NOs: 23 to 25, and the VL includes CDRs of SEQ ID NOs: 26, 27, and 10. MUC1 antibody or antigen binding fragment.
[17]
[1] or [2] above, wherein the VH includes the complementarity determining regions (CDRs) of SEQ ID NOs: 28, 29, and 25, and the VL includes the CDRs of SEQ ID NOs: 30, 9, and 31. An anti-glycoMUC1 antibody or antigen-binding fragment of.
[18]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [1] or [2] above, which is a chimeric antibody or a humanized antibody.
[19]
[1] or [2] above, wherein the VH comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 3, and the VL comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 4. 2] anti-glycoMUC1 antibody or antigen-binding fragment.
[20]
[1] or [2] above, wherein the VH comprises an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 3, and the VL comprises an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 4. 2] anti-glycoMUC1 antibody or antigen-binding fragment.
[21]
[1] or [2] above, wherein the VH comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 3, and the VL comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 4. 2] anti-glycoMUC1 antibody or antigen-binding fragment.
[22]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [1] or [2] above, wherein the VH includes the amino acid sequence of SEQ ID NO: 3, and the VL includes the amino acid sequence of SEQ ID NO: 4.
[23]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [1] or [2] above, which is multivalent.
[24]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [1] or [2] above, which is in the form of a single chain variable fragment (scFv).
[25]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [24] above, wherein the scFv comprises a heavy chain variable fragment on the N-terminal side of a light chain variable fragment.
[26]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [24] above, wherein the heavy chain variable fragment and light chain variable fragment of the scFv are covalently bonded to a linker sequence of 4 to 15 amino acids.
[27]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [1] or [2] above, which is in the form of a multispecific antibody.
[28]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [27] above, wherein the multispecific antibody is a bispecific antibody that binds to a second epitope different from the first epitope.
[29]
The anti-glycosylation antibody according to [28] above, wherein the bispecific antibody is a CrossMab, a Fab arm exchanged antibody, a bispecific T cell derivative (BiTE), or a biaffinity retargeting molecule (DART). MUC1 antibody or antigen binding fragment.
[30]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [29] above, wherein the bispecific antibody is CrossMab.
[31]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [30] above, wherein the bispecific antibody is CrossMab ^FAB .
[32]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [30] above, wherein the bispecific antibody is CrossMab ^VH-VL .
[33]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [30] above, wherein the bispecific antibody is CrossMab ^CH1-CL .
[34]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [29] above, wherein the bispecific antibody is a Fab arm exchanged antibody.
[35]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [29] above, wherein the bispecific antibody is a dual affinity retargeting molecule (DART).
[36]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [29] above, wherein the bispecific antibody is a bispecific T cell derivative (BiTE).
[37]
The anti-glyco MUC1 antibody or antigen-binding fragment according to [28] above, wherein the second epitope is a MUC1 epitope.
[38]
The anti-glyco MUC1 antibody or antigen-binding fragment according to [28] above, wherein the second epitope is a MUC1 epitope that is overexpressed on cancer cells compared to normal cells.
[39]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [28] above, wherein the second epitope is a T cell epitope.
[40]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [39] above, wherein the T cell epitope includes a CD3 epitope, a CD8 epitope, a CD16 epitope, a CD25 epitope, a CD28 epitope, or an NKG2D epitope.
[41]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [40] above, wherein the T cell epitope includes a CD3 epitope, and the CD3 epitope is optionally an epitope present in human CD3.
[42]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [41] above, wherein the CD3 epitope includes a CD3 gamma epitope, a CD3 delta epitope, a CD3 epsilon epitope, or a CD3 zeta epitope.
[43]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [1] or [2] above, which is conjugated to a detectable moiety.
[44]
The anti-glycoMUC1 antibody or antigen-binding fragment according to [43] above, wherein the detectable marker is an enzyme, a radioisotope, or a fluorescent label.
[45]
A fusion protein comprising the amino acid sequence of the anti-glycoMUC1 antibody or antigen-binding fragment according to [1] or [2] above, operably linked to at least a second amino acid sequence.
[46]
The fusion protein according to [45] above, wherein the second amino acid sequence is an amino acid sequence of 4-1BB, CD3-zeta, or a fragment thereof.
[47]
The fusion protein according to [45] above, wherein the second amino acid sequence is an amino acid sequence of a fusion peptide.
[48]
The fusion protein according to [47] above, wherein the fusion peptide is a CD28-CD3-zeta fusion peptide or a 4-1BB(CD137)-CD3-zeta fusion peptide.
[49]
The fusion protein according to [45] above, wherein the second amino acid sequence is an amino acid sequence of a modulator of T cell activation or a fragment thereof.
[50]
The fusion protein according to [49] above, wherein the modulator of T cell activation is IL-15 or IL-15Ra.
[51]
A chimeric antigen receptor (CAR) comprising the scFv described in [24] above.
[52]
The CAR according to [51] above, comprising, in order from the amino terminus to the carboxy terminus, a human CD8 leader peptide, the scFv, a human CD8 hinge domain, a human CD8 transmembrane domain, and a CD3-zeta signaling domain.
[53]
An antibody-drug conjugate comprising the anti-glycoMUC1 antibody or antigen-binding fragment according to [1] or [2] above, conjugated to a cytotoxic substance.
[54]
The cytotoxic substance is described in [53] above, wherein the cytotoxic substance is auristatin, a DNA minor groove binder, an alkylating agent, enediyne, lexitropsin, duocarmycin, a taxane, dolastatin, maytansinoid, or vinca alkaloid. antibody-drug conjugate.
[55]
The antibody-drug conjugate according to [54] above, wherein the anti-glycoMUC1 antibody or antigen-binding fragment is conjugated to the cytotoxic substance via a linker.
[56]
The antibody-drug conjugate according to the above [55], wherein the linker is cleavable under intracellular conditions.
[57]
The antibody-drug conjugate according to [56] above, wherein the cleavable linker is cleavable by an intracellular protease.
[58]
The antibody-drug conjugate according to [57] above, wherein the linker comprises a dipeptide.
[59]
The antibody-drug conjugate according to [58] above, wherein the dipeptide is val-cit or phe-lys.
[60]
The antibody-drug conjugate according to [56] above, wherein the cleavable linker is hydrolyzable at a pH of less than 5.5.
[61]
The antibody-drug conjugate according to [60] above, wherein the hydrolyzable linker is a hydrazone linker.
[62]
The antibody-drug conjugate according to [56] above, wherein the cleavable linker is a disulfide linker.
[63]
A nucleic acid comprising the coding region of the anti-glycoMUC1 antibody or antigen-binding fragment according to [1] or [2] above.
[64]
The nucleic acid according to [63] above, wherein the coding region is codon-optimized for expression in human cells.
[65]
A vector comprising the nucleic acid according to [63] above.
[66]
The vector described in [65] above, which is a viral vector.
[67]
The vector according to [66] above, wherein the viral vector is a lentiviral vector.
[68]
A host cell engineered to express the nucleic acid according to [63] above.
[69]
A host cell that is a human T cell engineered to express the CAR described in [51] above.
[70]
A host cell containing the vector described in [65] above.
[71]
A host cell that is a T cell containing the vector encoding the CAR according to [51] above.
[72]
(a) the anti-glycoMUC1 antibody or antigen-binding fragment according to [1] or [2] above, and
(b) a physiologically suitable buffer, adjuvant or diluent;
A pharmaceutical composition comprising.
[73]
A method of treating cancer, the method comprising administering an effective amount of the anti-glycoMUC1 antibody or antigen-binding fragment according to [1] or [2] above to a subject in need thereof.
[74]
The method according to [73] above, wherein the subject has breast cancer, non-small cell lung cancer, prostate cancer, pancreatic cancer, esophageal cancer, or colorectal cancer.
[75]
A method for detecting cancer in a biological sample, the method comprising: contacting the sample with the anti-glyco MUC1 antibody or antigen-binding fragment according to [1] or [2] above; and the anti-glyco MUC1 antibody or antigen. A method comprising detecting binding of bound fragments.
[76]
The method according to [75] above, further comprising quantifying binding of the anti-glycoMUC1 antibody or antigen-binding fragment.
[77]
The method according to [75] above, wherein said binding is compared to a normal tissue control as a negative/baseline control and/or compared to a cancerous tissue control as a positive control.

8. References

Claims (44)

An anti-glycoMUC1 antibody or antigen-binding fragment thereof ,
(i) Complementarity determining region (CDR) H1 containing the amino acid sequence of SEQ ID NO: 5 , CDR-H2 containing the amino acid sequence of SEQ ID NO: 29, CDR-H3 containing the amino acid sequence of SEQ ID NO: 25, amino acid sequence of SEQ ID NO: 8 CDR-L1 comprising, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR -L3 comprising the amino acid sequence of SEQ ID NO: 31,
(ii) CDR-H1 containing the amino acid sequence of SEQ ID NO: 23, CDR-H2 containing the amino acid sequence of SEQ ID NO: 29, CDR-H3 containing the amino acid sequence of SEQ ID NO: 25, CDR-L1 containing the amino acid sequence of SEQ ID NO: 8. , CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 31,
(iii) CDR-H1 containing the amino acid sequence of SEQ ID NO: 28, CDR-H2 containing the amino acid sequence of SEQ ID NO: 29, CDR-H3 containing the amino acid sequence of SEQ ID NO: 25, CDR-L1 containing the amino acid sequence of SEQ ID NO: 8. , CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 31, or
(iv) CDR-H1 containing the amino acid sequence of SEQ ID NO: 32, CDR-H2 containing the amino acid sequence of SEQ ID NO: 29, CDR-H3 containing the amino acid sequence of SEQ ID NO: 25, CDR-L1 containing the amino acid sequence of SEQ ID NO: 8. , CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 31,
Anti-glycoMUC1 antibody or antigen-binding fragment thereof . a. GalNAc-T1, GalNAc-T2, and GalNAc are purified recombinant human glycosyltransferases that preferentially bind to the glycoMUC1 epitope that is overexpressed on cancer cells compared to normal cells. - binds to MUC1 tandem repeat (VTSAPDTRPAPGSTAPPAHG) ₃ (SEQ ID NO: 47 ) glycosylated in vitro using T4; and
b. compete with an antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) sequence of SEQ ID NO: 3 and a light chain variable (VL) sequence of SEQ ID NO: 4 for binding to breast cancer cell lines MCF7 or T47D;
The anti-glycoMUC1 antibody or antigen-binding fragment thereof according to claim 1. (i) CDR-H1 contains the amino acid sequence of SEQ ID NO: 5 ,
CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6 ,
CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7 ,
CDR-L1 comprises the amino acid sequence of SEQ ID NO: 26 ,
CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27 , and
CDR-L3 contains the amino acid sequence of SEQ ID NO: 10 ,
(ii) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 5,
CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6,
CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7,
CDR-L1 comprises the amino acid sequence of SEQ ID NO: 30,
CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27, and
CDR-L3 contains the amino acid sequence of SEQ ID NO: 10,
(iii) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 5,
CDR-H2 comprises the amino acid sequence of SEQ ID NO: 24,
CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7,
CDR-L1 comprises the amino acid sequence of SEQ ID NO: 26,
CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27, and
CDR-L3 contains the amino acid sequence of SEQ ID NO: 10,
(iv) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 5,
CDR-H2 comprises the amino acid sequence of SEQ ID NO: 24,
CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7,
CDR-L1 comprises the amino acid sequence of SEQ ID NO: 30,
CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27, and
CDR-L3 contains the amino acid sequence of SEQ ID NO: 10,
(v) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 23,
CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6,
CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7,
CDR-L1 comprises the amino acid sequence of SEQ ID NO: 26,
CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27, and
CDR-L3 contains the amino acid sequence of SEQ ID NO: 10,
(vi) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 23,
CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6,
CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7,
CDR-L1 comprises the amino acid sequence of SEQ ID NO: 30,
CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27, and
CDR-L3 contains the amino acid sequence of SEQ ID NO: 10,
(vii) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 23,
CDR-H2 comprises the amino acid sequence of SEQ ID NO: 24,
CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7,
CDR-L1 comprises the amino acid sequence of SEQ ID NO: 26,
CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27, and
CDR-L3 contains the amino acid sequence of SEQ ID NO: 10,
(viii) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 23,
CDR-H2 comprises the amino acid sequence of SEQ ID NO: 24,
CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7,
CDR-L1 comprises the amino acid sequence of SEQ ID NO: 30,
CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27, and
CDR-L3 contains the amino acid sequence of SEQ ID NO: 10,
(ix) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 28,
CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6,
CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7,
CDR-L1 comprises the amino acid sequence of SEQ ID NO: 26,
CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27, and
CDR-L3 contains the amino acid sequence of SEQ ID NO: 10,
(x) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 28,
CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6,
CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7,
CDR-L1 comprises the amino acid sequence of SEQ ID NO: 30,
CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27, and
CDR-L3 contains the amino acid sequence of SEQ ID NO: 10,
(xi) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 28,
CDR-H2 comprises the amino acid sequence of SEQ ID NO: 24,
CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7,
CDR-L1 comprises the amino acid sequence of SEQ ID NO: 26,
CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27, and
CDR-L3 contains the amino acid sequence of SEQ ID NO: 10,
(xii) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 28,
CDR-H2 comprises the amino acid sequence of SEQ ID NO: 24,
CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7,
CDR-L1 comprises the amino acid sequence of SEQ ID NO: 30,
CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27, and
CDR-L3 contains the amino acid sequence of SEQ ID NO: 10,
(xiii) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 32,
CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6,
CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7,
CDR-L1 comprises the amino acid sequence of SEQ ID NO: 26,
CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27, and
CDR-L3 contains the amino acid sequence of SEQ ID NO: 10,
(xiv) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 32,
CDR-H2 comprises the amino acid sequence of SEQ ID NO: 6,
CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7,
CDR-L1 comprises the amino acid sequence of SEQ ID NO: 30,
CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27, and
CDR-L3 contains the amino acid sequence of SEQ ID NO: 10,
(xv) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 32,
CDR-H2 comprises the amino acid sequence of SEQ ID NO: 24,
CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7,
CDR-L1 comprises the amino acid sequence of SEQ ID NO: 26,
CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27, and
CDR-L3 comprises the amino acid sequence of SEQ ID NO: 10, or
(xvi) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 32,
CDR-H2 comprises the amino acid sequence of SEQ ID NO: 24,
CDR-H3 comprises the amino acid sequence of SEQ ID NO: 7,
CDR-L1 comprises the amino acid sequence of SEQ ID NO: 30,
CDR-L2 comprises the amino acid sequence of SEQ ID NO: 27, and
CDR-L3 comprises the amino acid sequence of SEQ ID NO: 10,
The anti-glycoMUC1 antibody or antigen-binding fragment thereof according to claim 1 or 2. (i) VH containing complementarity determining region (CDR) H1 containing the amino acid sequence of SEQ ID NO: 5, CDR-H2 containing the amino acid sequence of SEQ ID NO: 6, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 7, and SEQ ID NO: 8 or _
(ii) VH containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 23, CDR-H2 containing the amino acid sequence of SEQ ID NO: 24, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 25, and the amino acid sequence of SEQ ID NO: 26. a VL comprising CDR-L1 , CDR-L2 comprising the amino acid sequence of SEQ ID NO: 27, and CDR-L3 comprising the amino acid sequence of SEQ ID NO : 10; or
(iii) VH containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 28, CDR-H2 containing the amino acid sequence of SEQ ID NO: 29, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 25, and the amino acid sequence of SEQ ID NO: 30. A VL comprising CDR-L1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 31.
The anti-glycoMUC1 antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising: a VH comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3 and a VL comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 4; or at least 97% sequence identity to SEQ ID NO: 3. a VH comprising an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 4; or a VH comprising an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 3; or a VH comprising an amino acid sequence having 100% sequence identity with SEQ ID NO: 3 and an amino acid sequence having 100% sequence identity with SEQ ID NO: 4; including VL
The anti-glycoMUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising: The anti-glycoMUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which is a chimeric antibody or a humanized antibody. a. is polyvalent; or b. in the form of multispecific antibodies,
The anti-glycoMUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6. Anti-glycoMUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, in the form of a single chain variable fragment (scFv). i. said scFv comprises a heavy chain variable fragment N-terminal to a light chain variable fragment; and/or ii. 9. The anti-glycoMUC1 antibody or antigen-binding fragment thereof according to claim 8, wherein the scFv heavy chain variable fragment and light chain variable fragment are covalently linked to a linker sequence of 4 to 15 amino acids. The anti-glycoMUC1 antibody or antigen-binding fragment thereof is in the form of a multispecific antibody, and the multispecific antibody is directed against a glycoMUC1 epitope (first epitope) and a second epitope different from the first epitope. The anti-glycoMUC1 antibody or antigen-binding fragment thereof according to claim 7, which is a binding bispecific antibody. i. said bispecific antibody is a CrossMab, a Fab arm swapped antibody, a bispecific T cell derivative (BiTE), or a biaffinity retargeting molecule (DART); and/or ii. The second epitope is
(a) is a MUC1 epitope; or (b) is a T cell epitope.
The anti-glycoMUC1 antibody or antigen-binding fragment thereof according to claim 10. The anti-glycoMUC1 antibody or antigen-binding fragment thereof according to claim 11, wherein the bispecific antibody is CrossMab, and the CrossMab is CrossMab ^FAB , CrossMab ^VH-VL , or CrossMab ^CH1-CL . 13. The anti-glyco-MUC1 antibody or antigen-binding fragment thereof according to claim 11 or 12, wherein the second epitope is a MUC1 epitope that is overexpressed on cancer cells compared to normal cells. The anti-glycoMUC1 antibody or antigen-binding fragment thereof according to claim 11 or 12, wherein the second epitope is a T cell epitope including a CD3 epitope, a CD8 epitope, a CD16 epitope, a CD25 epitope, a CD28 epitope, or an NKG2D epitope. . 15. The anti-glycoMUC1 antibody or antigen-binding fragment thereof according to claim 14, wherein the CD3 epitope is an epitope present in human CD3. 16. The anti-glycoMUC1 antibody or antigen-binding fragment thereof according to claim 15, wherein the CD3 epitope is a CD3 gamma epitope, a CD3 delta epitope, a CD3 epsilon epitope, or a CD3 zeta epitope. An anti-glycoMUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, conjugated to a detectable moiety. 18. The anti-glycoMUCl antibody or antigen-binding fragment thereof of claim 17, wherein the detectable moiety is an enzyme, a radioisotope, or a fluorescent label. A fusion protein comprising the amino acid sequence of an anti-glycoMUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 operably linked to at least a second amino acid sequence. The second amino acid sequence is
a. is the amino acid sequence of 4-1BB, CD3-zeta, or a fragment thereof; or b. is the amino acid sequence of the fusion peptide; or c. is an amino acid sequence of a modulator of T cell activation or a fragment thereof;
A fusion protein according to claim 19. The fusion protein according to claim 20, wherein the fusion peptide is a CD28-CD3-zeta fusion peptide or a 4-1BB(CD137)-CD3-zeta fusion peptide. 21. The second amino acid sequence is an amino acid sequence of a modulator of T cell activation or a fragment thereof, and the modulator of T cell activation is IL-15 or IL-15Ra. fusion protein. A chimeric antigen receptor (CAR) comprising the anti-glycoMUC1 antibody or antigen-binding fragment thereof according to claim 8 or 9. 24. The CAR of claim 23, comprising, in order from amino-terminus to carboxy-terminus, a human CD8 leader peptide, the scFv, a human CD8 hinge domain, a human CD8 transmembrane domain, and a CD3-zeta signaling domain. An antibody-drug conjugate comprising the anti-glycoMUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 conjugated to a cytotoxic substance. a. The cytotoxic substance is an auristatin, a DNA minor groove binder, an alkylating agent, an enediyne, a lexitropsin, a duocarmycin, a taxane, a dolastatin, a maytansinoid, or a vinca alkaloid; and/or b. 26. The antibody-drug conjugate of claim 25, wherein the anti-glycoMUC1 antibody or antigen-binding fragment thereof is conjugated to the cytotoxic agent via a linker. 27. The antibody-drug conjugate of claim 26, wherein the anti-glycoMUC1 antibody or antigen-binding fragment thereof is conjugated to the cytotoxic agent via a linker that is cleavable under intracellular conditions. i. the linker is cleavable by an intracellular protease; or
ii. the linker is hydrolyzable at a pH below 5.5; or iii. the linker is a disulfide linker;
The antibody-drug conjugate of claim 27. 29. The antibody-drug conjugate of claim 28, wherein the linker is cleavable by an intracellular protease and comprises a dipeptide. 30. The antibody-drug conjugate of claim 29, wherein the dipeptide is val-cit or phe-lys. 29. The antibody-drug conjugate of claim 28, wherein the linker is hydrolyzable at a pH below 5.5 and is a hydrazone linker. The coding region of an anti-glycoMUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, the coding region of a fusion protein according to any one of claims 19 to 22, or the coding region of a fusion protein according to any one of claims 19 to 22, or A nucleic acid comprising the coding region of CAR according to 24. 33. The nucleic acid of claim 32, wherein the coding region is codon optimized for expression in human cells. A vector comprising the nucleic acid according to claim 32 or 33. 35. The vector of claim 34, which is a viral vector. 36. The vector of claim 35, wherein the viral vector is a lentiviral vector. A host cell engineered to express a nucleic acid according to claim 32 or 33, or comprising a vector according to any one of claims 34 to 36. 38. The host cell of claim 37, which is a human T cell engineered to express the CAR of claim 23 or 24. (a) the anti-glycoMUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, the fusion protein according to any one of claims 19 to 22, the fusion protein according to any one of claims 23 or 24; CAR, an antibody-drug conjugate according to any one of claims 25-31, a nucleic acid according to claims 32 or 33, a vector according to any one of claims 34-36, or claim 37 or 38, and (b) a pharmaceutical composition comprising a physiologically suitable buffer, adjuvant or diluent. 40. A pharmaceutical composition according to claim 39 for treating cancer. 41. The pharmaceutical composition according to claim 40, wherein the cancer is breast cancer, non-small cell lung cancer, prostate cancer, pancreatic cancer, esophageal cancer, or colorectal cancer. A method for detecting the presence or absence of cancer cells expressing glycoMUC1 in a biological sample, the method comprising:
a. contacting a biological sample with an anti-glycoMUC1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18; and b. A method comprising detecting binding of an anti-glycoMUC1 antibody or antigen-binding fragment thereof . 43. The method of claim 42, further comprising quantifying binding of the anti-glycoMUC1 antibody or antigen binding fragment thereof . 44. The method of claim 42 or 43, further comprising comparing the binding to a normal tissue control as a negative/baseline control and/or to a cancerous tissue control as a positive control.